Afucosylated anti-fgfr2iiib antibodies

ABSTRACT

The present invention provides antibodies that bind FGFR2IIIb, wherein the antibodies are afucosylated. The present invention provides compositions comprising antibodies that bind FGFR2IIIb, wherein at least 95% of the antibodies in the composition are afucosylated. In some embodiments, methods of treating cancer comprising administering afucosylated anti-FGFR2IIIb antibodies are provided.

This application is a Divisional of U.S. patent application Ser. No.14/447,751, filed Jul. 31, 2014, which claims the benefit of priority toU.S. Provisional Application No. 61/861,198, filed Aug. 1, 2013;61/901,732, filed Nov. 8, 2013; and 61/933,632, filed Jan. 30, 2014; thedisclosure of each of which is incorporated herein by reference in itsentirety for any purpose.

FIELD OF THE INVENTION

Afucosylated anti-FGFR2IIIb antibodies are provided.

BACKGROUND OF THE INVENTION

The fibroblast growth factor (FGF) family members bind to four knowntyrosine kinase receptors, fibroblast growth factor receptors 1-4(FGFR1-4) and their isoforms, with the various FGFs binding thedifferent FGFRs to varying extents (Zhang et al., J. Biol. Chem.281:15694, 2006). A protein sequence of human FGFR2 is provided in,e.g., GenBank Locus AF487553. Each FGFR consists of an extracellulardomain (ECD) comprising three immunoglobulin (Ig)-like domains (D1, D2and D3), a single transmembrane helix, and an intracellular catalytickinase domain (Mohammadi et al., Cytokine Growth Factor Revs, 16:107,2005). There is a contiguous stretch of acidic amino acids in the linkerbetween D1 and D2 called the “acid box” (AB). The region containing D1and AB is believed to be involved in autoinhibition of the receptor,which is relieved by binding to ligand. FGFs bind to the receptorsprimarily through regions in D2 and D3 of the receptors. The FGFRs arecharacterized by multiple alternative splicing of their mRNAs, leadingto a variety of isoforms (Ornitz et al., J. Biol. Chem. 271:15292, 1996;see also Swiss-Prot P21802 and isoforms P21802-1 to -20 for sequences ofFGFR2 and its isoforms). Notably, there are forms containing all threeIg domains (a isoform) or only the two Ig domains D2 and D3 domainswithout D1 (β isoform). In FGFR1-FGFR3, all forms contain the first halfof D3 denoted Ma, but two alternative exons can be utilized for thesecond half of D3, leading to IIIb and Mc forms. For FGFR2, these arerespectively denoted FGFR2IIIb and FGFR2IIIc (or just FGFR2b andFGFR2c); the corresponding beta forms are denoted FGFR2(beta)IIIb andFGFR2(beta)IIIc. The FGFR2IIIb form of FGFR2 (also denoted K-sam-II) isa high affinity receptor for both FGF1 and KGF family members (FGF7,FGF10, and FGF22) whereas FGFR2IIIc (also denoted K-sam-I) binds bothFGF1 and FGF2 well but does not bind the KGF family members (Miki etal., Proc. Natl. Acad. Sci. USA 89:246, 1992). Indeed, FGFR2IIIb is theonly receptor for KGF family members (Ornitz et al., 1996, op. cit.) andis therefore also designated KGFR.

The FGFRs and their isoforms are differentially expressed in varioustissues. FGFR2IIIb (and the IIIb forms of FGFR1 and FGFR3) is expressedin epithelial tissues, while FGFRIIIc is expressed in mesenchymaltissues (Duan et al., J. Biol. Chem. 267:16076, 1992; Ornitz et al.,1996, op. cit.). Certain of the FGF ligands of these receptors have anopposite pattern of expression. Thus, KGF subfamily members, includingFGF7 (KGF), FGF10, and FGF22, bind only to FGFRIIIb (Zhang et al., op.cit.) and are expressed in mesenchymal tissues so may be paracrineeffectors of epithelial cells (Ornitz et al., 1996, op. cit.). Incontrast, the FGF4 subfamily members FGF4-6 bind to FGFR2IIIc and areexpressed in both epithelial and mesenchymal lineages so may have eitherautocrine or paracrine functions. Because of the expression patterns ofthe isoforms of FGFR2 and their ligands, FGFR2 plays a role inepithelial-mesynchymal interactions (Finch et al., Dev. Dyn. 203:223,1995), so it is not surprising that knock-out of FGFR2IIIb in mice leadsto severe embryonic defects and lethality (De Moerlooze et al.,Development 127:483, 2000).

KGF (FGF7) and KGFR (FGFR2IIIb) are overexpressed in many pancreaticcancers (Ishiwata et al., Am. J. Pathol. 153: 213, 1998), and theircoexpression correlates with poor prognosis (Cho et al., Am. J. Pathol.170:1964, 2007). Somatic mutations of the FGFR2 gene were found in 12%of a large panel of endometrial (uterine) carcinomas, and in severaltested cases were required for tumor cell survival (Dutt et al., Proc.Natl. Acad. Sci. USA 105:8713, 2008). In two tumors the FGFR2 mutationwas found to be the same S252W substitution associated with Apertsyndrome. Amplification and overexpression of FGFR2 is associated withthe undifferentiated, diffuse type of gastric cancer, which has aparticularly poor prognosis, and inhibition of the FGFR2 activity bysmall molecule compounds potently inhibited proliferation of such cancercells (Kunii et al., Cancer Res. 68:2340, 2008; Nakamura et al.,Gastroenterol. 131:1530, 2006).

SUMMARY OF THE INVENTION

In some embodiments, an anti-FGFR2IIIb antibody is provided, wherein theheavy chain variable region comprises: (i) HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 6; (ii) HVR-H2 comprising the amino acidsequence of SEQ ID NO: 7; and (iii) HVR-H3 comprising the amino acidsequence of SEQ ID NO: 8; and the light chain variable region comprises:(iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (v)HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (vi)HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11; wherein theantibody is afucosylated. In some embodiments, the antibody lack fucoseat Asn297. In some embodiments, compositions comprising a plurality ofanti-FGFR2IIIb antibodies are provided, wherein the heavy chain variableregion of each anti-FGFR2IIIb antibody in the composition comprises: (i)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (ii) HVR-H2comprising the amino acid sequence of SEQ ID NO: 7; and (iii) HVR-H3comprising the amino acid sequence of SEQ ID NO: 8; and the light chainvariable region of each anti-FGFR2IIIb antibody in the compositioncomprises: (iv) HVR-L1 comprising the amino acid sequence of SEQ ID NO:9; (v) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and(vi) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11; whereinat least 95% of the antibodies in the composition are afucosylated. Insome embodiments, the composition may be a supernatant from anantibody-producing cell line. In some embodiments, the composition maybe a buffered composition. In some embodiments, the heavy chain variabledomain comprises the amino acid sequence of SEQ ID NO: 4. In someembodiments, the light chain variable domain comprises the amino acidsequence of SEQ ID NO: 5. In some embodiments, the heavy chain variabledomain comprises the amino acid sequence of SEQ ID NO: 4 and the lightchain variable domain comprises the amino acid sequence of SEQ ID NO: 5.In some embodiments, the heavy chain comprises the amino acid sequenceof SEQ ID NO: 2. In some embodiments, the light chain comprises theamino acid sequence of SEQ ID NO: 3. In some embodiments, the heavychain comprises the amino acid sequence of SEQ ID NO: 2 and the lightchain comprises the amino acid sequence of SEQ ID NO: 3. In someembodiments, a composition comprising a plurality of afucosylatedanti-FGFR2IIIb antibodies is provided, wherein the antibodies competefor binding to FGFR2IIIb with an antibody comprising a heavy chainvariable domain comprising the amino acid sequence of SEQ ID NO: 4 and alight chain variable domain comprising the amino acid sequence of SEQ IDNO: 5.

In some embodiments, the antibodies are monoclonal antibodies. In someembodiments, the antibodies are chimeric antibodies. In someembodiments, the antibodies are humanized antibodies. In any of theembodiments described herein, the antibodies may comprise a κ lightchain constant region. In any of the embodiments described herein, theantibodies may comprise an IgG1 heavy chain constant region.

In some embodiments, the antibodies have enhanced ADCC activity in vitrocompared to fucosylated anti-FGFR2IIIb antibodies having the same aminoacid sequence. In some embodiments, the afucosylated anti-FGFR2IIIbantibodies cause specific lysis that is at least 10, at least 15, atleast 20, at least 25, at least 30, at least 35, at least 40, at least45, at least 50, at least 60, at least 65, at least 70, or at least 75percentage points greater than specific lysis with fucosylatedanti-FGFR2IIIb antibodies. In some embodiments, ADCC activity isdetermined using Ba/F3 cells expressing FGFR2IIIb as target cells andisolated human PBMCs as effector cells.

In some embodiments, the antibodies have enhanced affinity for Fc gammaRIIIA compared to fucosylated anti-FGFR2IIIb antibodies having the sameamino acid sequence. In some embodiments, the afucosylatedanti-FGFR2IIIb antibodies bind to Fc gamma RIIIA with at least 2-fold,at least 3-fold, at least 4-fold, at least 5-fold, at least 7-fold, atleast 10-fold, at least 12-fold, at least 15-fold, at least 17-fold, orat least 20-fold greater affinity than fucosylated anti-FGFR2IIIbantibodies. In some embodiments, affinity for Fc gamma RIIIA isdetermined using surface plasmon resonance. In some embodiments, Fcgamma RIIIA is selected from Fc gamma RIIIA(V158) and Fc gammaRIIIA(F158). In some embodiments, Fc gamma RIIIA is Fc gammaRIIIA(V158).

In any of the embodiments described herein, the antibodies may bindFGFR2IIIb but not FGFR2IIIc.

In any of the embodiments described herein, a composition comprising aplurality of afucosylated anti-FGFR2IIIb antibodies comprises at least95% afucosylated antibodies. In any of the embodiments described herein,a composition comprising a plurality of afucosylated anti-FGFR2IIIbantibodies may have undetectable fucosylation. In some embodiments, thepresence of fucose may be determined by a method comprising highperformance liquid chromatography (HPLC), capillary electrophoresis, orMALDI-TOF mass spectrometry.

In some embodiments, host cells are provided that comprise nucleic acidencoding an anti-FGFR2IIIb antibody described herein, wherein the hostcell lacks a functional alpha-1,6-fucosyltransferase gene (FUT8) gene.In some embodiments, the host cell is a CHO cell.

In some embodiments, methods for making afucosylated anti-FGFR2IIIbantibodies are provided. In some embodiments, a method comprisesculturing a host cell under conditions suitable for expressing nucleicacid encoding the anti-FGFR2IIIb antibody, wherein the host cell lacks afunctional alpha-1,6-fucosyltransferase gene (FUT8) gene. In someembodiments, a method for making afucosylated anti-FGFR2IIIb antibodiescomprises culturing the host cell under conditions suitable forproducing the afucosylated anti-FGFR2IIIb antibody. In some embodiments,the method further comprises recovering the anti-FGFR2IIIb antibodyproduced by the host cell. In some embodiments, less than 5% of theanti-FGFR2IIIb antibodies produced by the host cell comprise fucose. Insome embodiments, at least 95% of the anti-FGFR2IIIb antibodies producedby the host cell lack fucose (i.e., are afucosylated). In someembodiments, fucose is undetectable in the anti-FGFR2IIIb antibodiesproduced by the host cell. In some embodiments, presence of fucose isdetected by a method comprising HPLC, capillary electrophoresis, orMALDI-TOF mass spectrometry.

In some embodiments, pharmaceutical compositions are provided, whereinthe pharmaceutical composition comprises afucosylated anti-FGFR2IIIbantibodies described herein and a pharmaceutically acceptable carrier.

In some embodiments, methods of treating cancer are provided. In someembodiments, a method comprises administering an effective amount of apharmaceutical composition comprising afucosylated anti-FGFR2IIIbantibodies described herein and a pharmaceutically acceptable carrier.In some embodiments, the cancer is selected from gastric cancer, breastcancer, ovarian cancer, endometrial cancer, pancreatic cancer, andesophageal cancer. In some embodiments, the cancer is gastric cancer. Insome embodiments, the cancer comprises an FGFR2 gene amplification. Insome embodiments, FGFR2 amplification comprises FGFR2:CEN10 (chromosome10 centromere) ratio of >3. In some embodiments, the canceroverexpresses FGFR2IIIb. In some embodiments, a cancer comprising FGFR2amplification overexpresses FGFR2IIIb to a greater extent thanFGFR2IIIc. In some embodiments, a cancer comprising FGFR2 amplificationexpresses FGFR2IIIb at a normalized level that is more than 2-fold,3-fold, 5-fold, or 10-fold greater than the normalized level ofFGFR2IIIc expression. In some embodiments, the expression levels arenormalized to GUSB. In some embodiments, the cancer overexpressesFGFR2IIIb but does not comprise FGFR2 gene amplification. In someembodiments, expression or overexpression of FGFR2IIIb is determined byIHC. In some embodiments, 1+, 2+ or 3+ staining of tumor cells by IHCindicates FGFR2IIIb overexpression. In some embodiments, 2+ or 3+staining in tumor cells by IHC indicates FGFR2IIIb overexpression. Insome embodiments, the IHC staining is scored as described in Example 6.

In some embodiments, a method of treating cancer further comprisesadministering at least one additional therapeutic agent selected from aplatinum agent, paclitaxel, ABRAXANE®, docetaxel, gemcitabine,capecitabine, irinotecan, epirubicin, FOLFOX, FOLFIRI, leucovorin,fluorouracil, mitomycin C, and doxorubicin hydrochloride. In someembodiments, the platinum agent is selected from cisplatin, oxaliplatin,and carboplatin. In some embodiments, a method of treating cancerfurther comprises administering paclitaxel. In some embodiments, amethod of treating cancer further comprises administering cisplatinand/or 5-FU.

In some embodiments, uses of a pharmaceutical composition comprisingafucosylated anti-FGFR2IIIb antibodies described herein and apharmaceutically acceptable carrier are provided. In some embodiments,such use is for treating cancer in an individual with cancer. In someembodiments, the cancer is selected from gastric cancer, breast cancer,ovarian cancer, endometrial cancer, pancreatic cancer, or esophagealcancer. In some embodiments, the cancer is gastric cancer. In someembodiments, the cancer comprises an FGFR2 gene amplification. In someembodiments, FGFR2 amplification comprises FGFR2:CEN10 (chromosome 10centromere) ratio of >3. In some embodiments, the cancer overexpressesFGFR2IIIb. In some embodiments, a cancer comprising FGFR2 amplificationoverexpresses FGFR2IIIb to a greater extent than FGFR2IIIc. In someembodiments, a cancer comprising FGFR2 amplification expresses FGFR2IIIbat a normalized level that is more than 2-fold, 3-fold, 5-fold, or10-fold greater than the normalized level of FGFR2IIIc expression. Insome embodiments, the expression levels are normalized to GUSB. In someembodiments, the cancer overexpresses FGFR2IIIb but does not compriseFGFR2 gene amplification. In some embodiments, expression oroverexpression of FGFR2IIIb is determined by IHC. In some embodiments,1+, 2+ or 3+ staining of tumor cells by IHC indicates FGFR2IIIboverexpression. In some embodiments, 2+ or 3+ staining in tumor cells byIHC indicates FGFR2IIIb overexpression. In some embodiments, the IHCstaining is scored as described in Example 6.

In some embodiments, pharmaceutical compositions for treating cancer areprovided, wherein the pharmaceutical composition comprises afucosylatedanti-FGFR2IIIb antibodies described herein and a pharmaceuticallyacceptable carrier. In some embodiments, the cancer is selected fromgastric cancer, breast cancer, ovarian cancer, endometrial cancer,pancreatic cancer, or esophageal cancer. In some embodiments, the canceris gastric cancer. In some embodiments, the cancer comprises an FGFR2gene amplification. In some embodiments, FGFR2 amplification comprisesFGFR2:CEN10 (chromosome 10 centromere) ratio of >3. In some embodiments,the cancer overexpresses FGFR2IIIb. In some embodiments, a cancercomprising FGFR2 amplification overexpresses FGFR2IIIb to a greaterextent than FGFR2IIIc. In some embodiments, a cancer comprising FGFR2amplification expresses FGFR2IIIb at a normalized level that is morethan 2-fold, 3-fold, 5-fold, or 10-fold greater than the normalizedlevel of FGFR2IIIc expression. In some embodiments, the expressionlevels are normalized to GUSB. In some embodiments, the canceroverexpresses FGFR2IIIb but does not comprise FGFR2 gene amplification.In some embodiments, expression or overexpression of FGFR2IIIb isdetermined by IHC. In some embodiments, 1+, 2+ or 3+ staining of tumorcells by IHC indicates FGFR2IIIb overexpression. In some embodiments, 2+or 3+ staining in tumor cells by IHC indicates FGFR2IIIb overexpression.In some embodiments, the IHC staining is scored as described in Example6.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1C show ADCC activity of afucosylated αFGFR2bA andfucosylated αFGFR2bF against FGFR2IIIb-expressing Ba/F3 cells, asdiscussed in Example 3. In the legends, “αFGFR2bF/FGFR2b” indicates thatfucosylated αFGFR2bF antibody was tested against FGFR2IIIb-expressingBa/F3 target cells.

FIGS. 2A to 2D show efficacy of afucosylated αFGFR2bA and fucosylatedαFGFR2bF in an OCUM-2M gastric cancer xenograft model, at (A and B) 10mg/kg and (C and D) 3 mg/kg, as discussed in Example 4.

FIGS. 3A and 3B show dose-dependent efficacy of afucosylated αFGFR2bA inan OCUM-2M gastric cancer xenograft model, as discussed in Example 4.

FIGS. 4A and 4B show efficacy of combination therapy with afucosylatedαFGFR2bA and paclitaxel in an OCUM-2M gastric cancer xenograft model, asdiscussed in Example 4.

FIGS. 5A and 5B show efficacy of combination therapy with afucosylatedαFGFR2bA and 5-FU/cisplatin in an OCUM-2M gastric cancer xenograftmodel, as discussed in Example 4.

FIGS. 6A and 6B show efficacy of afucosylated αFGFR2bA in a MFM-223breast cancer xenograft model, as discussed in Example 4.

FIGS. 7A and 7B show the glycan profile of αFGFR2b antibody produced in(7A) Potelligent® CHOK1SV cells and (7B) CHOK1SV cells, as described inExample 1.

FIG. 8 shows schematic diagrams of N-linked glycans typically found inantibodies.

FIG. 9 shows ADCC of Ba/F3 FGF2b cells with increasing concentrations ofαFGFR2bA or αFGFR2bF. Assays were performed with normal human PBMC at anE:T ratio of 25:1, as described in Example 5. Data are plotted as LDHrelease.

FIG. 10 shows ADCC of OCUM-2M cells with increasing concentrations ofαFGFR2bA or αFGFR2bF. Assays were performed with normal human PBMC at anE:T ratio of 25:1. As described in Example 5. Data are plotted aspercent specific lysis.

FIGS. 11A to 11F show detection of FGFR2IIIb in tumor tissue samplesusing immunohistochemistry, as described in Example 6.

DETAILED DESCRIPTION OF THE INVENTION

Afucosylated antibodies that bind FGFR2IIIb are provided. In someembodiments, afucosylated antibody heavy chains and light chains thatare capable of forming antibodies that bind FGFR2IIIb are also provided.In some embodiments, afucosylated antibodies, heavy chains, and lightchains comprising one or more particular hypervariable regions (HVRs)are provided. In some embodiments, afucosylated anti-FGFR2IIIbantibodies have enhanced ADCC activity relative to fucosylatedanti-FGFR2IIIb antibodies. In some embodiments, afucosylatedanti-FGFR2IIIb antibodies have enhanced affinity for Fc gamma RIIIArelative to fucosylated anti-FGFR2IIIb antibodies. In some embodiments,afucosylated anti-FGFR2IIIb antibodies have enhanced affinity for Fcgamma RIIIA(V158) relative to fucosylated anti-FGFR2IIIb antibodies. Insome embodiments, afucosylated anti-FGFR2IIIb antibodies have enhancedaffinity for Fc gamma RIIIA(F158) relative to fucosylated anti-FGFR2IIIbantibodies. In some embodiments, afucosylated anti-FGFR2IIIb antibodiesdo not bind to FGFR2IIIc.

Polynucleotides encoding antibodies that bind FGFR2IIIb are provided.Polynucleotides encoding antibody heavy chains or lights chains are alsoprovided. Host cells that express afucosylated anti-FGFR2IIIb antibodiesare provided. Methods of treatment using afucosylated antibodies toFGFR2IIIb are provided. Such methods include, but are not limited to,methods of treating cancer, such as gastric cancer, breast cancer,ovarian cancer, endometrial cancer, pancreatic cancer, and esophagealcancer.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

All references cited herein, including patent applications, patentpublications, and Genbank Accession numbers are herein incorporated byreference, as if each individual reference were specifically andindividually indicated to be incorporated by reference in its entirety.

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized methodologies described in Sambrook et al., MolecularCloning: A Laboratory Manual 3rd. edition (2001) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. CURRENT PROTOCOLS INMOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (2003)); the seriesMETHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICALAPPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)),Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMALCELL CULTURE (R. I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; CellBiology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press;Animal Cell Culture (R. I. Freshney), ed., 1987); Introduction to Celland Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press;Cell and Tissue Culture Laboratory Procedures (A. Doyle, J. B.Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Handbookof Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); GeneTransfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos,eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds.,1994); Current Protocols in Immunology (J. E. Coligan et al., eds.,1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRLPress, 1988-1989); Monoclonal Antibodies: A Practical Approach (P.Shepherd and C. Dean, eds., Oxford University Press, 2000); UsingAntibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold SpringHarbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D.Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principlesand Practice of Oncology (V. T. DeVita et al., eds., J.B. LippincottCompany, 1993); and updated versions thereof.

I. DEFINITIONS

Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context or expressly indicated, singularterms shall include pluralities and plural terms shall include thesingular.

It is understood that aspect and embodiments of the invention describedherein include “consisting” and/or “consisting essentially of” aspectsand embodiments. As used herein, the singular form “a”, “an”, and “the”includes plural references unless indicated otherwise.

In this application, the use of “or” means “and/or” unless expresslystated or understood by one skilled in the art. In the context of amultiple dependent claim, the use of “or” refers back to more than onepreceding independent or dependent claim.

As is understood by one skilled in the art, reference to “about” a valueor parameter herein includes (and describes) embodiments that aredirected to that value or parameter per se. For example, descriptionreferring to “about X” includes description of “X”.

The terms “nucleic acid molecule”, “nucleic acid” and “polynucleotide”may be used interchangeably, and refer to a polymer of nucleotides. Suchpolymers of nucleotides may contain natural and/or non-naturalnucleotides, and include, but are not limited to, DNA, RNA, and PNA.“Nucleic acid sequence” refers to the linear sequence of nucleotidesthat comprise the nucleic acid molecule or polynucleotide.

The terms “polypeptide” and “protein” are used interchangeably to referto a polymer of amino acid residues, and are not limited to a minimumlength. Such polymers of amino acid residues may contain natural ornon-natural amino acid residues, and include, but are not limited to,peptides, oligopeptides, dimers, trimers, and multimers of amino acidresidues. Both full-length proteins and fragments thereof areencompassed by the definition. The terms also include post-expressionmodifications of the polypeptide, for example, glycosylation,sialylation, acetylation, phosphorylation, and the like. Furthermore,for purposes of the present invention, a “polypeptide” refers to aprotein which includes modifications, such as deletions, additions, andsubstitutions (generally conservative in nature), to the nativesequence, as long as the protein maintains the desired activity. Thesemodifications may be deliberate, as through site-directed mutagenesis,or may be accidental, such as through mutations of hosts which producethe proteins or errors due to PCR amplification.

“FGFR2IIIb” or “FGFR2b” are used interchangeably to refer to thefibroblast growth factor receptor 2 IIIb splice form. An exemplary humanFGFR2IIIb is shown in GenBank Accession No. NP_075259.4, dated Jul. 7,2013. A nonlimiting exemplary mature human FGFR2IIIb amino acid sequenceis shown in SEQ ID NO: 1.

“FGFR2IIIc” or “FGFR2c” are used interchangeably to refer to thefibroblast growth factor receptor 2 Mc splice form. An exemplary humanFGFR2IIIc is shown in GenBank Accession No. NP_000132.3, dated Jul. 7,2013. A nonlimiting exemplary mature FGFR2IIIc amino acid sequence isshown in SEQ ID NO: 12.

The term “epitope” refers to a site on a target molecule (e.g., anantigen, such as a protein, nucleic acid, carbohydrate or lipid) towhich an antigen-binding molecule (e.g., an antibody, antibody fragment,or scaffold protein containing antibody binding regions binds. Epitopesoften consist of a chemically active surface grouping of molecules suchas amino acids, polypeptides or sugar side chains and have specificthree-dimensional structural characteristics as well as specific chargecharacteristics. Epitopes can be formed both from contiguous orjuxtaposed noncontiguous residues (e.g., amino acids, nucleotides,sugars, lipid moiety) of the target molecule. Epitopes formed fromcontiguous residues (e.g., amino acids, nucleotides, sugars, lipidmoiety) typically are retained on exposure to denaturing solventswhereas epitopes formed by tertiary folding typically are lost ontreatment with denaturing solvents. An epitope may include but notlimited to at least 3, at least 5 or 8-10 residues (e.g., amino acids ornucleotides). In some examples an epitope is less than 20 residues(e.g., amino acids or nucleotides) in length, less than 15 residues orless than 12 residues. Two antibodies may bind the same epitope withinan antigen if they exhibit competitive binding for the antigen.

A “nonlinear epitope” or “conformational epitope” comprisesnoncontiguous polypeptides, amino acids and/or sugars within theantigenic protein to which an antibody specific to the epitope binds.

A “linear epitope” comprises contiguous polypeptides, amino acids and/orsugars within the antigenic protein to which an antibody specific to theepitope binds.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

The term antibody includes, but is not limited to, fragments that arecapable of binding antigen, such as Fv, single-chain Fv (scFv), Fab,Fab′, and (Fab′)₂. Papain digestion of antibodies produces two identicalantigen-binding fragments, called “Fab” fragments, each with a singleantigen-binding site, and a residual “Fc” fragment, whose name reflectsits ability to crystallize readily. Pepsin treatment yields an F(ab′)₂fragment that has two antigen-combining sites and is still capable ofcross-linking antigen. The term antibody also includes, but is notlimited to, chimeric antibodies, humanized antibodies, and antibodies ofvarious species such as mouse, human, cynomolgus monkey, etc.

The term “heavy chain variable region” refers to a region comprisingheavy chain HVR1, framework (FR) 2, HVR2, FR3, and HVR3. In someembodiments, a heavy chain variable region also comprises at least aportion of an FR1 and/or at least a portion of an FR4.

The term “heavy chain constant region” refers to a region comprising atleast three heavy chain constant domains, C_(H)1, C_(H)2, and C_(H)3.Nonlimiting exemplary heavy chain constant regions include γ, δ, and α.Nonlimiting exemplary heavy chain constant regions also include and μ.Each heavy constant region corresponds to an antibody isotype. Forexample, an antibody comprising a γ constant region is an IgG antibody,an antibody comprising a δ constant region is an IgD antibody, and anantibody comprising an α constant region is an IgA antibody. Further, anantibody comprising μ constant region is an IgM antibody, and anantibody comprising an ε constant region is an IgE antibody. Certainisotypes can be further subdivided into subclasses. For example, IgGantibodies include, but are not limited to, IgG1 (comprising a γ₁constant region), IgG2 (comprising a γ₂ constant region), IgG3(comprising a γ₃ constant region), and IgG4 (comprising a γ₄ constantregion) antibodies; IgA antibodies include, but are not limited to, IgA1(comprising an α₁ constant region) and IgA2 (comprising an α₂ constantregion) antibodies; and IgM antibodies include, but are not limited to,IgM1 and IgM2.

The term “heavy chain” refers to a polypeptide comprising at least aheavy chain variable region, with or without a leader sequence. In someembodiments, a heavy chain comprises at least a portion of a heavy chainconstant region. The term “full-length heavy chain” refers to apolypeptide comprising a heavy chain variable region and a heavy chainconstant region, with or without a leader sequence.

The term “light chain variable region” refers to a region comprisinglight chain HVR1, framework (FR) 2, HVR2, FR3, and HVR3. In someembodiments, a light chain variable region also comprises an FR1 and/oran FR4.

The term “light chain constant region” refers to a region comprising alight chain constant domain, C_(L). Nonlimiting exemplary light chainconstant regions include λ, and κ.

The term “light chain” refers to a polypeptide comprising at least alight chain variable region, with or without a leader sequence. In someembodiments, a light chain comprises at least a portion of a light chainconstant region. The term “full-length light chain” refers to apolypeptide comprising a light chain variable region and a light chainconstant region, with or without a leader sequence.

The term “hypervariable region” or “HVR” refers to each of the regionsof an antibody variable domain which are hypervariable in sequenceand/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theV_(H) (H1, H2, H3), and three in the V_(L) (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe “complementarity determining regions” (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.Exemplary hypervariable loops occur at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3).(Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs(CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acidresidues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 ofH2, and 95-102 of H3. (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)). The terms hypervariableregions (HVRs) and complementarity determining regions (CDRs), are usedherein interchangeably in reference to portions of the variable regionthat form the antigen binding regions.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain(V_(L)) framework or a heavy chain variable domain (V_(H)) frameworkderived from a human immunoglobulin framework or a human consensusframework, as defined below. An acceptor human framework derived from ahuman immunoglobulin framework or a human consensus framework maycomprise the same amino acid sequence thereof, or it may contain aminoacid sequence changes. In some embodiments, the number of amino acidchanges are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 orless, 4 or less, 3 or less, or 2 or less. In some embodiments, the V_(L)acceptor human framework is identical in sequence to the V_(L) humanimmunoglobulin framework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). In someembodiments, “binding affinity” refers to intrinsic binding affinitywhich reflects a 1:1 interaction between members of a binding pair(e.g., antibody and antigen). The affinity of a molecule X for itspartner Y can generally be represented by the dissociation constant(K_(d)). Affinity can be measured by common methods known in the art,including those described herein.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs) and/orcomplementarity determining regions (CDRs), compared to a parentantibody which does not possess such alterations, such alterationsresulting in an improvement in the affinity of the antibody for antigen.

A “chimeric antibody” refers to an antibody in which a portion of theheavy and/or light chain is derived from a particular source or species,while the remainder of the heavy and/or light chain is derived from adifferent source or species. In some embodiments, a chimeric antibodyrefers to an antibody comprising at least one variable region from afirst species (such as mouse, rat, cynomolgus monkey, etc.) and at leastone constant region from a second species (such as human, cynomolgusmonkey, etc.). In some embodiments, a chimeric antibody comprises atleast one mouse variable region and at least one human constant region.In some embodiments, a chimeric antibody comprises at least onecynomolgus variable region and at least one human constant region. Insome embodiments, all of the variable regions of a chimeric antibody arefrom a first species and all of the constant regions of the chimericantibody are from a second species.

A “humanized antibody” refers to an antibody in which at least one aminoacid in a framework region of a non-human variable region has beenreplaced with the corresponding amino acid from a human variable region.In some embodiments, a humanized antibody comprises at least one humanconstant region or fragment thereof. In some embodiments, a humanizedantibody is an Fab, an scFv, a (Fab′)₂, etc.

An “HVR-grafted antibody” refers to a humanized antibody in which one ormore hypervariable regions (HVRs) of a first (non-human) species havebeen grafted onto the framework regions (FRs) of a second (human)species.

A “functional Fc region” possesses an “effector function” of a nativesequence Fc region. Exemplary “effector functions” include Fc receptorbinding; C1q binding; CDC; ADCC; phagocytosis; down regulation of cellsurface receptors (e.g. B cell receptor; BCR), etc. Such effectorfunctions generally require the Fc region to be combined with a bindingdomain (e.g., an antibody variable domain) and can be assessed usingvarious assays.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. Nativesequence human Fc regions include a native sequence human IgG1 Fc region(non-A and A allotypes); native sequence human IgG2 Fc region; nativesequence human IgG3 Fc region; and native sequence human IgG4 Fc regionas well as naturally occurring variants thereof.

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one aminoacid modification.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. In some embodiments, an FcγR is a native human FcR. Insome embodiments, an FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof those receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domainInhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see, e.g., Daeron,Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example,in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126:330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein.

The term “Fc receptor” or “FcR” also includes the neonatal receptor,FcRn, which is responsible for the transfer of maternal IgGs to thefetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J.Immunol. 24:249 (1994)) and regulation of homeostasis ofimmunoglobulins. Methods of measuring binding to FcRn are known (see,e.g., Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie etal., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol.Chem. 279(8):6213-6216 (2004); WO 2004/92219 (Hinton et al.).

“Effector functions” refer to biological activities attributable to theFc region of an antibody, which vary with the antibody isotype. Examplesof antibody effector functions include: C1q binding and complementdependent cytotoxicity (CDC); Fc receptor binding; antibody-dependentcell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cellsurface receptors (e.g. B cell receptor); and B cell activation.

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. In certain embodiments, the cells express atleast FcγRIII and perform ADCC effector function(s). Examples of humanleukocytes which mediate ADCC include peripheral blood mononuclear cells(PBMC), natural killer (NK) cells, monocytes, macrophages, cytotoxic Tcells, and neutrophils. The effector cells may be isolated from a nativesource, e.g., from blood.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g. NK cells, neutrophils, andmacrophages) enable these cytotoxic effector cells to bind specificallyto an antigen-bearing target cell and subsequently kill the target cellwith cytotoxins. The primary cells for mediating ADCC, NK cells, expressFcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII FcRexpression on hematopoietic cells is summarized in Table 3 on page 464of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess ADCCactivity of a molecule of interest, an in vitro ADCC assay, such as thatdescribed in U.S. Pat. No. 5,500,362 or 5,821,337 or U.S. Pat. No.6,737,056 (Presta), may be performed. Useful effector cells for suchassays include PBMC and NK cells. Alternatively, or additionally, ADCCactivity of the molecule of interest may be assessed in vivo, e.g., inan animal model such as that disclosed in Clynes et al. Proc. Natl.Acad. Sci. (USA) 95:652-656 (1998). Additional antibodies with alteredFc region amino acid sequences and increased or decreased ADCC activityare described, e.g., in U.S. Pat. No. 7,923,538, and U.S. Pat. No.7,994,290.

An antibody having an “enhanced ADCC activity” refers to an antibodythat is more effective at mediating ADCC in vitro or in vivo compared tothe parent antibody, wherein the antibody and the parent antibody differin at least one structural aspect, and when the amounts of such antibodyand parent antibody used in the assay are essentially the same. In someembodiments, the antibody and the parent antibody have the same aminoacid sequence, but the antibody is afucosylated while the parentantibody is fucosylated. In some embodiments, ADCC activity will bedetermined using the in vitro ADCC assay as herein disclosed, but otherassays or methods for determining ADCC activity, e.g. in an animal modeletc., are contemplated. In some embodiments, an antibody with enhancedADCC activity has enhanced affinity for Fc gamma RIIIA In someembodiments, an antibody with enhanced ADCC activity has enhancedaffinity for Fc gamma RIIIA (V158). In some embodiments, an antibodywith enhanced ADCC activity has enhanced affinity for Fc gamma RIIIA(F158).

An antibody with “altered” FcR binding affinity or ADCC activity is onewhich has either enhanced or diminished FcR binding activity and/or ADCCactivity compared to a parent antibody, wherein the antibody and theparent antibody differ in at least one structural aspect. An antibodythat “displays increased binding” to an FcR binds at least one FcR withbetter affinity than the parent antibody. An antibody that “displaysdecreased binding” to an FcR, binds at least one FcR with lower affinitythan a parent antibody. Such antibodies that display decreased bindingto an FcR may possess little or no appreciable binding to an FcR, e.g.,0-20% binding to the FcR compared to a native sequence IgG Fc region.

“Enhanced affinity for Fc gamma RIIIA” refers to an antibody that hasgreater affinity for Fc gamma RIIIA (also referred to, in someinstances, as CD16a) than a parent antibody, wherein the antibody andthe parent antibody differ in at least one structural aspect. In someembodiments, the antibody and the parent antibody have the same aminoacid sequence, but the antibody is afucosylated while the parentantibody is fucosylated. Any suitable method for determining affinityfor Fc gamma RIIIA may be used. In some embodiments, affinity for Fcgamma RIIIA is determined by a method described herein. In someembodiments, an antibody with enhanced affinity for Fc gamma RIIIA hasenhanced ADCC activity. In some embodiments, an antibody with enhancedaffinity for Fc gamma RIIIA has enhanced affinity for Fc gammaRIIIA(V158). In some embodiments, an antibody with enhanced affinity forFc gamma RIIIA has enhanced affinity for Fc gamma RIIIA(F158).

“Afucosylated” antibody or an antibody “lacking fucose” refers to anIgG1 or IgG3 isotype antibody that lacks fucose in its constant regionglycosylation. Glycosylation of human IgG1 or IgG3 occurs at Asn297 ascore fucosylated biantennary complex oligosaccharide glycosylationterminated with up to 2 Gal residues. In some embodiments, anafucosylated antibody lacks fucose at Asn297. These structures aredesignated as G0, G1 (α1,6 or α1,3) or G2 glycan residues, depending onthe amount of terminal Gal residues. See, e.g., Raju, T. S., BioProcessInt. 1: 44-53 (2003). CHO type glycosylation of antibody Fc isdescribed, e.g., in Routier, F. H., Glycoconjugate J. 14: 201-207(1997). In some embodiments, at least 85% of a batch of antibodiesrecombinantly expressed in non glycomodified CHO host cells arefucosylated at Asn297. When referring to a composition comprising aplurality of antibodies, the antibodies are considered to beafucosylated if <5% of the antibodies in the composition comprise fucoseat Asn297. Methods of measuring fucose include any methods known in theart, including the methods described herein. In some embodiments, fucoseis detected by the method described in Example 1. In some embodiments,fucose is undetectable in a composition comprising a plurality ofafucosylated antibodies. In some embodiments, an afucosylated antibodyhas enhanced ADCC activity. In some embodiments, an afucosylatedantibody has enhanced affinity for Fc gamma RIIIA In some embodiments,an afucosylated antibody has enhanced affinity for Fc gamma RIIIA(V158).In some embodiments, an afucosylated antibody has enhanced affinity forFc gamma RIIIA(F158).

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass),which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g., as described in Gazzano-Santoro et al.,J. Immunol. Methods 202:163 (1996), may be performed. Antibodies withaltered Fc region amino acid sequences and increased or decreased C1qbinding capability are described, e.g., in U.S. Pat. No. 6,194,551 B1,U.S. Pat. No. 7,923,538, U.S. Pat. No. 7,994,290 and WO 1999/51642. Seealso, e.g., Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

The term “substantially similar” or “substantially the same,” as usedherein, denotes a sufficiently high degree of similarity between two ormore numeric values such that one of skill in the art would consider thedifference between the two or more values to be of little or nobiological and/or statistical significance within the context of thebiological characteristic measured by said value. In some embodimentsthe two or more substantially similar values differ by no more thanabout any one of 5%, 10%, 15%, 20%, 25%, or 50%.

The phrase “substantially reduced,” or “substantially different,” asused herein, denotes a sufficiently high degree of difference betweentwo numeric values such that one of skill in the art would consider thedifference between the two values to be of statistical significancewithin the context of the biological characteristic measured by saidvalues. In some embodiments, the two substantially different numericvalues differ by greater than about any one of 10%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%.

The terms “leader sequence” and “signal sequence” are usedinterchangeably to refer to a sequence of amino acid residues located atthe N terminus of a polypeptide that facilitates secretion of apolypeptide from a mammalian cell. A leader sequence may be cleaved uponexport of the polypeptide from the mammalian cell, forming a matureprotein. Leader sequences may be natural or synthetic, and they may beheterologous or homologous to the protein to which they are attached.

A “native sequence” polypeptide comprises a polypeptide having the sameamino acid sequence as a polypeptide found in nature. Thus, a nativesequence polypeptide can have the amino acid sequence of naturallyoccurring polypeptide from any mammal. Such native sequence polypeptidecan be isolated from nature or can be produced by recombinant orsynthetic means. The term “native sequence” polypeptide specificallyencompasses naturally occurring truncated or secreted forms of thepolypeptide (e.g., an extracellular domain sequence), naturallyoccurring variant forms (e.g., alternatively spliced forms) andnaturally occurring allelic variants of the polypeptide.

A polypeptide “variant” means a biologically active polypeptide havingat least about 80% amino acid sequence identity with the native sequencepolypeptide after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Such variants include, for instance, polypeptides wherein oneor more amino acid residues are added, or deleted, at the N- orC-terminus of the polypeptide. In some embodiments, a variant will haveat least about 80% amino acid sequence identity. In some embodiment, avariant will have at least about 90% amino acid sequence identity. nsome embodiment, a variant will have at least about 95% amino acidsequence identity with the native sequence polypeptide.

As used herein, “Percent (%) amino acid sequence identity” and“homology” with respect to a peptide, polypeptide or antibody sequenceare defined as the percentage of amino acid residues in a candidatesequence that are identical with the amino acid residues in the specificpeptide or polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.

An amino acid substitution may include but are not limited to thereplacement of one amino acid in a polypeptide with another amino acid.Conservative substitutions are shown in Table 1 under the heading of“preferred substitutions”. More substantial changes are provided inTable 1 under the heading of “exemplary substitutions”, and as furtherdescribed below in reference to amino acid side chain classes. Aminoacid substitutions may be introduced into an antibody of interest andthe products screened for a desired activity, e.g., retained/improvedantigen binding, decreased immunogenicity, or improved ADCC or CDC.

TABLE 1 Preferred Original Residue Exemplary Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

The term “vector” is used to describe a polynucleotide that may beengineered to contain a cloned polynucleotide or polynucleotides thatmay be propagated in a host cell. A vector may include one or more ofthe following elements: an origin of replication, one or more regulatorysequences (such as, for example, promoters and/or enhancers) thatregulate the expression of the polypeptide of interest, and/or one ormore selectable marker genes (such as, for example, antibioticresistance genes and genes that may be used in colorimetric assays,e.g., β-galactosidase). The term “expression vector” refers to a vectorthat is used to express a polypeptide of interest in a host cell.

A “host cell” refers to a cell that may be or has been a recipient of avector or isolated polynucleotide. Host cells may be prokaryotic cellsor eukaryotic cells. Exemplary eukaryotic cells include mammalian cells,such as primate or non-primate animal cells; fungal cells, such asyeast; plant cells; and insect cells. Nonlimiting exemplary mammaliancells include, but are not limited to, NSO cells, PER.C6® cells(Crucell), and 293 and CHO cells, and their derivatives, such as 293-6Eand DG44 cells, respectively.

The term “isolated” refers to a molecule that has been separated from atleast some of the components with which it is typically found in natureor produced. For example, a polypeptide is referred to as “isolated”when it is separated from at least some of the components of the cell inwhich it was produced. Where a polypeptide is secreted by a cell afterexpression, physically separating the supernatant containing thepolypeptide from the cell that produced it is considered to be“isolating” the polypeptide. Similarly, a polynucleotide is referred toas “isolated” when it is not part of the larger polynucleotide (such as,for example, genomic DNA or mitochondrial DNA, in the case of a DNApolynucleotide) in which it is typically found in nature, or isseparated from at least some of the components of the cell in which itwas produced, e.g., in the case of an RNA polynucleotide. Thus, a DNApolynucleotide that is contained in a vector inside a host cell may bereferred to as “isolated”.

The terms “individual” or “subject” are used interchangeably herein torefer to an animal; for example a mammal. In some embodiments, methodsof treating mammals, including, but not limited to, humans, rodents,simians, felines, canines, equines, bovines, porcines, ovines, caprines,mammalian laboratory animals, mammalian farm animals, mammalian sportanimals, and mammalian pets, are provided. In some examples, an“individual” or “subject” refers to an individual or subject in need oftreatment for a disease or disorder.

A “disease” or “disorder” refers to a condition where treatment isneeded.

The term “cancer” refers to a malignant proliferative disorderassociated with uncontrolled cell proliferation, unrestrained cellgrowth, and decreased cell death via apoptosis. Nonlimiting exemplarycancers include gastric cancer, breast cancer, ovarian cancer,endometrial cancer, pancreatic cancer, and esophageal cancer. In someembodiments, a cancer comprises an FGFR2 gene amplification. In someembodiments, FGFR2 amplification comprises FGFR2:CEN10 (chromosome 10centromere) ratio of >3. In some embodiments, a cancer comprising anFGFR2 gene amplification overexpresses FGFR2IIIb. In some embodiments, acancer comprising FGFR2 amplification overexpresses FGFR2IIIb to agreater extent than FGFR2IIIc. In some embodiments, a cancer comprisingFGFR2 amplification expresses FGFR2IIIb at a normalized level that ismore than 2-fold, 3-fold, 5-fold, or 10-fold greater than the normalizedlevel of FGFR2IIIc expression. In some embodiments, the expressionlevels are normalized to GUSB. In some embodiments, a canceroverexpresses FGFR2IIIb but does not comprise FGFR2 gene amplification.In some embodiments, a gastric cancer comprises an FGFR2 geneamplification. In some embodiments, a gastric cancer comprising an FGFR2gene amplification overexpresses FGFR2IIIb. In some embodiments, agastric cancer comprising FGFR2 amplification overexpresses FGFR2IIIb toa greater extent than FGFR2IIIc. In some embodiments, a gastric cancercomprising FGFR2 amplification expresses FGFR2IIIb at a normalized levelthat is more than 2-fold, 3-fold, 5-fold, or 10-fold greater than thenormalized level of FGFR2IIIc expression. In some embodiments, theexpression levels are normalized to GUSB. In some embodiments, a gastriccancer overexpresses FGFR2IIIb but does not comprise FGFR2 geneamplification. In some embodiments, overexpression is mRNAoverexpression. In some embodiments, overexpression is proteinoverexpression.

The term “tumor” is used herein to refer to a group of cells thatexhibit abnormally high levels of proliferation and growth. A tumor maybe benign, pre-malignant, or malignant; malignant tumor cells arecancerous. Tumor cells may be solid tumor cells or leukemic tumor cells.The term “tumor growth” is used herein to refer to proliferation orgrowth by a cell or cells that comprise a tumor that leads to acorresponding increase in the size of the tumor.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. “Treatment” as used herein, covers anyadministration or application of a therapeutic for disease in a mammal,including a human. For purposes of this invention, beneficial or desiredclinical results include, but are not limited to, any one or more of:alleviation of one or more symptoms, diminishment of extent of disease,preventing or delaying spread (e.g., metastasis, for example metastasisto the lung or to the lymph node) of disease, preventing or delayingrecurrence of disease, delay or slowing of disease progression,amelioration of the disease state, inhibiting the disease or progressionof the disease, inhibiting or slowing the disease or its progression,arresting its development, and remission (whether partial or total).Also encompassed by “treatment” is a reduction of pathologicalconsequence of a proliferative disease. The methods of the inventioncontemplate any one or more of these aspects of treatment.

In the context of cancer, the term “treating” includes any or all of:inhibiting growth of tumor cells or cancer cells, inhibiting replicationof tumor cells or cancer cells, lessening of overall tumor burden andameliorating one or more symptoms associated with the disease.

The terms “inhibition” or “inhibit” refer to a decrease or cessation ofany phenotypic characteristic or to the decrease or cessation in theincidence, degree, or likelihood of that characteristic. To “reduce” or“inhibit” is to decrease, reduce or arrest an activity, function, and/oramount as compared to a reference. In certain embodiments, by “reduce”or “inhibit” is meant the ability to cause an overall decrease of 20% orgreater. In another embodiment, by “reduce” or “inhibit” is meant theability to cause an overall decrease of 50% or greater. In yet anotherembodiment, by “reduce” or “inhibit” is meant the ability to cause anoverall decrease of 75%, 85%, 90%, 95%, or greater.

A “reference” as used herein, refers to any sample, standard, or levelthat is used for comparison purposes. A reference may be obtained from ahealthy and/or non-diseased sample. In some examples, a reference may beobtained from an untreated sample. In some examples, a reference isobtained from a non-diseased on non-treated sample of a subjectindividual. In some examples, a reference is obtained from one or morehealthy individuals who are not the subject or patient.

As used herein, “delaying development of a disease” means to defer,hinder, slow, retard, stabilize, suppress and/or postpone development ofthe disease (such as cancer). This delay can be of varying lengths oftime, depending on the history of the disease and/or individual beingtreated. As is evident to one skilled in the art, a sufficient orsignificant delay can, in effect, encompass prevention, in that theindividual does not develop the disease. For example, a late stagecancer, such as development of metastasis, may be delayed.

“Preventing,” as used herein, includes providing prophylaxis withrespect to the occurrence or recurrence of a disease in a subject thatmay be predisposed to the disease but has not yet been diagnosed withthe disease.

As used herein, to “suppress” a function or activity is to reduce thefunction or activity when compared to otherwise same conditions exceptfor a condition or parameter of interest, or alternatively, as comparedto another condition. For example, an antibody which suppresses tumorgrowth reduces the rate of growth of the tumor compared to the rate ofgrowth of the tumor in the absence of the antibody.

An “effective amount” of an agent refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic or prophylactic result.

A “therapeutically effective amount” of a substance/molecule of theinvention, agonist or antagonist may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the substance/molecule, agonist or antagonist to elicit adesired response in the individual. A therapeutically effective amountis also one in which any toxic or detrimental effects of thesubstance/molecule, agonist or antagonist are outweighed by thetherapeutically beneficial effects. A therapeutically effective amountmay be delivered in one or more administrations.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically but not necessarily, since a prophylacticdose is used in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

The terms “pharmaceutical formulation” and “pharmaceutical composition”refer to a preparation which is in such form as to permit the biologicalactivity of the active ingredient(s) to be effective, and which containsno additional components which are unacceptably toxic to a subject towhich the formulation would be administered. Such formulations may besterile.

A “pharmaceutically acceptable carrier” refers to a non-toxic solid,semisolid, or liquid filler, diluent, encapsulating material,formulation auxiliary, or carrier conventional in the art for use with atherapeutic agent that together comprise a “pharmaceutical composition”for administration to a subject. A pharmaceutically acceptable carrieris non-toxic to recipients at the dosages and concentrations employedand is compatible with other ingredients of the formulation. Thepharmaceutically acceptable carrier is appropriate for the formulationemployed.

A “sterile” formulation is aseptic or essentially free from livingmicroorganisms and their spores.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive or sequentialadministration in any order.

The term “concurrently” is used herein to refer to administration of twoor more therapeutic agents, where at least part of the administrationoverlaps in time or where the administration of one therapeutic agentfalls within a short period of time relative to administration of theother therapeutic agent. For example, the two or more therapeutic agentsare administered with a time separation of no more than about 60minutes, such as no more than about any of 30, 15, 10, 5, or 1 minutes.

The term “sequentially” is used herein to refer to administration of twoor more therapeutic agents where the administration of one or moreagent(s) continues after discontinuing the administration of one or moreother agent(s). For example, administration of the two or moretherapeutic agents are administered with a time separation of more thanabout 15 minutes, such as about any of 20, 30, 40, 50, or 60 minutes, 1day, 2 days, 3 days, 1 week, 2 weeks, or 1 month, or longer.

As used herein, “in conjunction with” refers to administration of onetreatment modality in addition to another treatment modality. As such,“in conjunction with” refers to administration of one treatment modalitybefore, during or after administration of the other treatment modalityto the individual.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

An “article of manufacture” is any manufacture (e.g., a package orcontainer) or kit comprising at least one reagent, e.g., a medicamentfor treatment of a disease or disorder (e.g., cancer), or a probe forspecifically detecting a biomarker described herein. In certainembodiments, the manufacture or kit is promoted, distributed, or sold asa unit for performing the methods described herein.

II. ANTI-FGFR2IIIB ANTIBODIES

In some aspects, the invention provides an afucosylated antibodydirected against FGFR2IIIb. Afucosylated anti-FGFR2IIIb antibodiesinclude, but are not limited to, humanized antibodies, chimericantibodies, mouse antibodies, and antibodies comprising the heavy chainand/or light chain HVRs (e.g. CDRs) discussed herein. In one aspect, theinvention provides isolated afucosylated antibodies that bind toFGFR2IIIb. In certain embodiments, an afucosylated anti-FGFR2IIIbantibody modulates FGFR2IIIb activity. In some embodiments, anafucosylated anti-FGFR2IIIb antibody has enhanced ADCC activity. In someembodiments, an afucosylated anti-FGFR2IIIb antibody has enhancedaffinity for Fc gamma RIIIA In some embodiments, an afucosylatedanti-FGFR2IIIb antibody has enhanced affinity for Fc gamma RIIIA(V158).In some embodiments, an afucosylated anti-FGFR2IIIb antibody hasenhanced affinity for Fc gamma RIIIA(F158).

The anti-FGFR2IIIb antibody designated “αFGFR2b” described herein in theExamples and the sequence listing is intended to have the same aminoacid sequence as antibody HuGAL-FR21 in U.S. Pat. No. 8,101,723 B2,issued Jan. 24, 2012. U.S. Pat. No. 8,101,723 B2 is specificallyincorporated herein by reference for any purpose, and in particular,FIGS. 13 and 14 of U.S. Pat. No. 8,101,723 B2, which show the amino acidsequences of the variable regions and full-length mature antibody chainsof HuGAL-FR21, are incorporated by reference herein for any purpose. Inaddition, the HVR sequences of antibody HuGAL-FR21, which are underlinedin FIG. 13 of U.S. Pat. No. 8,101,723 B2, are specifically incorporatedby reference herein for any purpose.

In one aspect, the invention provides an afucosylated anti-FGFR2IIIbantibody comprising at least one, two, three, four, five, or six HVRs(e.g., CDRs) selected from (a) HVR-H1 comprising the amino acid sequenceof SEQ ID NO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ IDNO: 7; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8;(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e)HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (f)HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

In some embodiments, an afucosylated anti-FGFR2IIIb antibody comprises aheavy chain variable region and a light chain variable region. In someembodiments, an afucosylated anti-FGFR2IIIb antibody comprises at leastone heavy chain comprising a heavy chain variable region and at least aportion of a heavy chain constant region, and at least one light chaincomprising a light chain variable region and at least a portion of alight chain constant region. In some embodiments, an afucosylatedanti-FGFR2IIIb antibody comprises two heavy chains, wherein each heavychain comprises a heavy chain variable region and at least a portion ofa heavy chain constant region, and two light chains, wherein each lightchain comprises a light chain variable region and at least a portion ofa light chain constant region. In some embodiments, an afucosylatedanti-FGFR2IIIb antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 4 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 5. Insome embodiments, an afucosylated anti-FGFR2IIIb antibody comprises aheavy chain comprising the amino acid sequence of SEQ ID NO: 2 and alight chain comprising the amino acid sequence of SEQ ID NO: 3.

In one aspect, the invention provides an afucosylated anti-FGFR2IIIbantibody comprising six HVRs comprising (a) HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 6; (b) HVR-H2 comprising the amino acidsequence of SEQ ID NO: 7; (c) HVR-H3 comprising the amino acid sequenceof SEQ ID NO: 8; (d) HVR-L1 comprising the amino acid sequence of SEQ IDNO: 9; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10;and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11. Insome embodiments, the afucosylated anti-FGFR2IIIb antibody comprises thesix HVRs as described above and binds to FGFR2IIIb. In some embodiments,the afucosylated anti-FGFR2IIIb antibody comprises the six HVRs asdescribed above, binds to FGFR2IIIb and has at least one activityselected from enhanced ADCC activity and enhanced affinity for Fc gammaRIIIA (such as Fc gamma RIIIA(V158) and/or Fc gamma RIIIA(F158)). Insome embodiments, the afucosylated anti-FGFRIIIb antibody does not bindto FGFR2IIIc.

In one aspect, the invention provides an afucosylated anti-FGFR2IIIbantibody that competes with an anti-FGFR2IIIb antibody comprising sixHVRs comprising (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7;(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8; (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 11.

In one aspect, the invention provides an afucosylated antibodycomprising at least one, at least two, or all three V_(H) HVR sequencesselected from (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7;and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8.

In another aspect, the invention provides an afucosylated antibodycomprising at least one, at least two, or all three V_(L) HVR sequencesselected from (a) HVR-L1 comprising the amino acid sequence of SEQ IDNO: 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10;and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

In another aspect, an afucosylated antibody of the invention comprises(a) a V_(H) domain comprising at least one, at least two, or all threeV_(H) HVR sequences selected from (i) HVR-H1 comprising the amino acidsequence of SEQ ID NO: 6, (ii) HVR-H2 comprising the amino acid sequenceof SEQ ID NO: 7, and (iii) HVR-H3 comprising an amino acid sequenceselected from SEQ ID NO: 8; and (b) a V_(L) domain comprising at leastone, at least two, or all three V_(L) HVR sequences selected from (i)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9, (ii) HVR-L2comprising the amino acid sequence of SEQ ID NO: 10, and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO: 11.

In another aspect, an afucosylated anti-FGFR2IIIb antibody comprises aheavy chain variable domain (V_(H)) sequence having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to theamino acid sequence of SEQ ID NO: 4. In certain embodiments, a V_(H)sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-FGFR2IIIb antibody comprising that sequence retains the ability tobind to FGFR2IIIb. In certain embodiments, such anti-FGFR2IIIb antibodyretains the ability to selectively bind to FGFR2IIIb without binding toFGFR2IIIc. In certain embodiments, a total of 1 to 10 amino acids havebeen substituted, inserted and/or deleted in SEQ ID NO: 4. In certainembodiments, substitutions, insertions, or deletions occur in regionsoutside the HVRs (i.e., in the FRs). Optionally, the afucosylatedanti-FGFR2IIIb antibody comprises the V_(H) sequence in SEQ ID NO: 5,including post-translational modifications of that sequence. In aparticular embodiment, the V_(H) comprises one, two or three HVRsselected from: (a) HVR-H1 comprising the amino acid sequence of SEQ IDNO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7;and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8.

In another aspect, an afucosylated anti-FGFR2IIIb antibody is provided,wherein the antibody comprises a light chain variable domain (V_(L))having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to the amino acid sequence of SEQ ID NO: 5. Incertain embodiments, a V_(L) sequence having at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions(e.g., conservative substitutions), insertions, or deletions relative tothe reference sequence, but an anti-FGFR2IIIb antibody comprising thatsequence retains the ability to bind to FGFR2IIIb. In certainembodiments, such anti-FGFR2IIIb antibody retains the ability toselectively bind to FGFR2IIIb without binding to FGFR2IIIc. In certainembodiments, a total of 1 to 10 amino acids have been substituted,inserted and/or deleted in SEQ ID NO: 5. In certain embodiments, thesubstitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the afucosylated anti-FGFR2IIIbantibody comprises the V_(L) sequence in SEQ ID NO: 4, includingpost-translational modifications of that sequence. In a particularembodiment, the V_(L) comprises one, two or three HVRs selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO: 10; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO: 11.

In another aspect, an afucosylated anti-FGFR2IIIb antibody comprises aheavy chain variable domain (V_(H)) sequence having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to theamino acid sequence of SEQ ID NO: 4 and a light chain variable domain(V_(L)) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:5. In certain embodiments, a V_(H) sequence having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence, and a V_(L) sequencehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-FGFR2IIIb antibody comprising that sequence retains the ability tobind to FGFR2IIIb. In certain embodiments, such anti-FGFR2IIIb antibodyretains the ability to selectively bind to FGFR2IIIb without binding toFGFR2IIIc. In certain embodiments, a total of 1 to 10 amino acids havebeen substituted, inserted and/or deleted in SEQ ID NO: 4. In certainembodiments, a total of 1 to 10 amino acids have been substituted,inserted and/or deleted in SEQ ID NO: 5. In certain embodiments,substitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the afucosylated anti-FGFR2IIIbantibody comprises the V_(H) sequence in SEQ ID NO: 4 and the V_(L)sequence of SEQ ID NO: 5, including post-translational modifications ofone or both sequence. In a particular embodiment, the V_(H) comprisesone, two or three HVRs selected from: (a) HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 6; (b) HVR-H2 comprising the amino acidsequence of SEQ ID NO: 7; and (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO: 8; and the V_(L) comprises one, two or three HVRsselected from (a) HVR-L1 comprising the amino acid sequence of SEQ IDNO: 9; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10;and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

In another aspect, an afucosylated anti-FGFR2IIIb antibody is provided,wherein the antibody comprises a V_(H) as in any of the embodimentsprovided above, and a V_(L) as in any of the embodiments provided above.In one embodiment, the antibody comprises the V_(H) and V_(L) sequencesin SEQ ID NO: 4 and SEQ ID NO: 5, respectively, includingpost-translational modifications of those sequences.

In another aspect, an afucosylated anti-FGFR2IIIb antibody comprises aheavy chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to the amino acid sequence ofSEQ ID NO: 2. In certain embodiments, a heavy chain sequence having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identitycontains substitutions (e.g., conservative substitutions), insertions,or deletions relative to the reference sequence, but an afucosylatedanti-FGFR2IIIb antibody comprising that sequence retains the ability tobind to FGFR2IIIb. In certain embodiments, such anti-FGFR2IIIb antibodyretains the ability to selectively bind to FGFR2IIIb without binding toFGFR2IIIc. In certain embodiments, a total of 1 to 10 amino acids havebeen substituted, inserted and/or deleted in SEQ ID NO: 2. In certainembodiments, substitutions, insertions, or deletions occur in regionsoutside the HVRs (i.e., in the FRs). Optionally, the afucosylatedanti-FGFR2IIIb antibody heavy chain comprises the V_(H) sequence in SEQID NO: 2, including post-translational modifications of that sequence.In a particular embodiment, the heavy chain comprises one, two or threeHVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQID NO: 6; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7;and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8.

In another aspect, an afucosylated anti-FGFR2IIIb antibody is provided,wherein the antibody comprises a light chain having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to theamino acid sequence of SEQ ID NO: 3. In certain embodiments, a lightchain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identity contains substitutions (e.g., conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-FGFR2IIIb antibody comprising that sequenceretains the ability to bind to FGFR2IIIb. In certain embodiments, suchanti-FGFR2IIIb antibody retains the ability to selectively bind toFGFR2IIIb without binding to FGFR2IIIc. In certain embodiments, a totalof 1 to 10 amino acids have been substituted, inserted and/or deleted inSEQ ID NO: 3. In certain embodiments, the substitutions, insertions, ordeletions occur in regions outside the HVRs (i.e., in the FRs).Optionally, the afucosylated anti-FGFR2IIIb antibody light chaincomprises the V_(L) sequence in SEQ ID NO: 3, includingpost-translational modifications of that sequence. In a particularembodiment, the light chain comprises one, two or three HVRs selectedfrom (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 9; (b)HVR-L2 comprising the amino acid sequence of SEQ ID NO: 10; and (c)HVR-L3 comprising the amino acid sequence of SEQ ID NO: 11.

In another aspect, an afucosylated anti-FGFR2IIIb antibody comprises aheavy chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to the amino acid sequence ofSEQ ID NO: 2 and a light chain sequence having at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to theamino acid sequence of SEQ ID NO: 3. In certain embodiments, a heavychain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identity contains substitutions (e.g., conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-FGFR2IIIb antibody comprising that sequenceretains the ability to bind to FGFR2IIIb. In certain embodiments, suchanti-FGFR2IIIb antibody retains the ability to selectively bind toFGFR2IIIb without binding to FGFR2IIIc. In certain embodiments, a lightchain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identity contains substitutions (e.g., conservativesubstitutions), insertions, or deletions relative to the referencesequence, but an anti-FGFR2IIIb antibody comprising that sequenceretains the ability to bind to FGFR2IIIb. In certain embodiments, suchanti-FGFR2IIIb antibody retains the ability to selectively bind toFGFR2IIIb without binding to FGFR2IIIc. In certain embodiments, a totalof 1 to 10 amino acids have been substituted, inserted and/or deleted inSEQ ID NO: 2. In certain embodiments, a total of 1 to 10 amino acidshave been substituted, inserted and/or deleted in SEQ ID NO: 3. Incertain embodiments, substitutions, insertions, or deletions occur inregions outside the HVRs (i.e., in the FRs). Optionally, theafucosylated anti-FGFR2IIIb antibody heavy chain comprises the V_(H)sequence in SEQ ID NO: 2, including post-translational modifications ofthat sequence and the afucosylated anti-FGFR2IIIb antibody light chaincomprises the V_(L) sequence in SEQ ID NO: 3, includingpost-translational modifications of that sequence. In a particularembodiment, the heavy chain comprises one, two or three HVRs selectedfrom: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6; (b)HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; and (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8; and the lightchain comprises one, two or three HVRs selected from (a) HVR-L1comprising the amino acid sequence of SEQ ID NO: 9; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO: 10; and (c) HVR-L3comprising the amino acid sequence of SEQ ID NO: 11.

Exemplary Chimeric Antibodies

In certain embodiments, an antibody, such as an afucosylated antibody,provided herein is a chimeric antibody. Certain chimeric antibodies aredescribed, e.g., in U.S. Pat. No. 4,816,567; and Morrison et al., (1984)Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In one example, achimeric antibody comprises a non-human variable region (e.g., avariable region derived from a mouse, rat, hamster, rabbit, or non-humanprimate, such as a monkey) and a human constant region. In a furtherexample, a chimeric antibody is a “class switched” antibody in which theclass or subclass has been changed from that of the parent antibody.Chimeric antibodies include antigen-binding fragments thereof.

Nonlimiting exemplary afucsoylated chimeric antibodies include chimericantibodies comprising heavy chain HVR1, HVR2, and HVR3, and/or lightchain HVR1, HVR2, and HVR3 sequences described herein. In someembodiments, the afucosylated chimeric anti-FGFR2IIIb antibody comprisesthe variable regions described above and binds to FGFR2IIIb. In someembodiments, the afucosylated chimeric anti-FGFR2IIIb antibody comprisesthe variable regions described above, binds to FGFR2IIIb and has atleast one activity selected from enhanced ADCC activity and enhancedaffinity for Fc gamma RIIIA (such as Fc gamma RIIIA(V158) and/or Fcgamma RIIIA(F158)). In some embodiments, the afucosylated chimericanti-FGFRIIIb antibody does not bind to FGFR2IIIc.

In some embodiments, an afucosylated chimeric anti-FGFR2IIIb antibodycomprises a heavy chain comprising a variable region sequence that is atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical SEQ ID NO: 4, wherein the antibody binds FGFR2IIIb. In someembodiments, an afucosylated chimeric anti-FGFR2IIIb antibody comprisesa light chain comprising a variable region sequence that is at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% identicalto SEQ ID NO: 5, wherein the antibody binds FGFR2IIIb. In someembodiments, an afucosylated chimeric anti-FGFR2IIIb antibody comprisesa heavy chain comprising a variable region sequence that is at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% identicalto SEQ ID NO: 4; and a light chain comprising a variable region sequencethat is at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identical to SEQ ID NO: 5; wherein the antibody binds FGFR2IIIb.

Exemplary afucosylated chimeric anti-FGFR2IIIb antibodies also includechimeric antibodies that compete for binding to FGFR2IIIb with anantibody or fragment thereof described herein. Thus, in someembodiments, a chimeric anti-FGFR2IIIb antibody is provided thatcompetes for binding to FGFR2IIIb with an antibody comprising a heavychain variable region comprising the amino acid sequence of SEQ ID NO: 4and a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 5. In some embodiments, the antibody competes for binding toFGFR2IIIb, but does not bind FGFR2IIIc.

In some embodiments, a chimeric antibody described herein comprises oneor more human constant regions. In some embodiments, the human heavychain constant region is of an isotype selected from IgA, IgG, and IgD.In some embodiments, the human light chain constant region is of anisotype selected from κ and λ. In some embodiments, a chimeric antibodydescribed herein comprises a human IgG constant region. In someembodiments, a chimeric antibody described herein comprises a human IgG4heavy chain constant region. In some embodiments, a chimeric antibodydescribed herein comprises a human IgG4 constant region and a human κlight chain.

As noted above, whether or not effector function is desirable may dependon the particular method of treatment intended for an antibody. Thus, insome embodiments, when effector function is desirable, a chimericanti-FGFR2IIIb antibody comprising a human IgG1 heavy chain constantregion or a human IgG3 heavy chain constant region is selected. In someembodiments, when effector function is not desirable, a chimericanti-FGFR2IIIb antibody comprising a human IgG4 or IgG2 heavy chainconstant region is selected.

Exemplary Humanized Antibodies

In some embodiments, afucosylated humanized antibodies that bindFGFR2IIIb are provided. Humanized antibodies are useful as therapeuticmolecules because humanized antibodies reduce or eliminate the humanimmune response to non-human antibodies (such as the human anti-mouseantibody (HAMA) response), which can result in an immune response to anantibody therapeutic, and decreased effectiveness of the therapeutic.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, (2008) Front. Biosci. 13: 1619-1633, and arefurther described, e.g., in Riechmann et al., (1988) Nature 332:323-329;Queen et al., (1989) Proc. Natl Acad. Sci. USA 86: 10029-10033; U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., (2005) Methods 36:25-34 (describing SDR (a-CDR) grafting); Padlan,(1991)Mol. Immunol. 28:489-498 (describing “resurfacing”); Dall'Acqua etal., (2005) Methods 36:43-60 (describing “FR shuffling”); and Osbourn etal., (2005) Methods 36:61-68 and Klimka et al., (2000) Br. J. Cancer,83:252-260 (describing the “guided selection” approach to FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. (1993) J. Immunol. 151:2296); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; and Presta etal. (1993) J. Immunol, 151:2623); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, (2008) Front. Biosci. 13:1619-1633); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al.,(1997) J. Biol. Chem. 272: 10678-10684 and Rosok et al., (1996) J. Biol.Chem. 271:22611-22618).

Nonlimiting exemplary humanized antibodies include αFGFR2b, describedherein. Nonlimiting exemplary afucosylated humanized antibodies includeαFGFR2bA, described herein, which has the same amino acid sequences asαFGFR2b comprising fucose (also referred to as αFGFR2bF). Nonlimitingexemplary afucosylated humanized antibodies also include antibodiescomprising a heavy chain variable region of αFGFR2b and/or a light chainvariable region of αFGFR2b. Nonlimiting exemplary afucosylated humanizedantibodies include antibodies comprising a heavy chain variable regionof SEQ ID NO: 4 and/or a light chain variable region of SEQ ID NO: 5.Exemplary humanized antibodies also include, but are not limited to,humanized antibodies comprising heavy chain HVR1, HVR2, and HVR3, and/orlight chain HVR1, HVR2, and HVR3 of αFGFR2b. In some embodiments, thehumanized anti-FGFR2IIIb antibody comprises the HVRs described above(i.e., (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 6;(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 7; (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 9; (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 10; and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 11) and binds toFGFR2IIIb. In some embodiments, the humanized anti-FGFR2IIIb antibodycomprises the HVRs described above, binds to FGFR2IIIb and has at leastone activity selected from enhanced ADCC activity and enhanced affinityfor Fc gamma RIIIA (such as Fc gamma RIIIA(V158) and/or Fc gammaRIIIA(F158)). In some embodiments, the afucosylated humanizedanti-FGFRIIIb antibody does not bind to FGFR2IIIc.

In some embodiments, an afucosylated humanized anti-FGFR2IIIb antibodycomprises heavy chain HVR1, HVR2, and HVR3 and/or a light chain HVR1,HVR2, and HVR3 of αFGFR2b. Nonlimiting exemplary afucosylated humanizedanti-FGFR2IIIb antibodies include antibodies comprising sets of heavychain HVR1, HVR2, and HVR3 set forth in SEQ ID NOs: 6, 7, and 8.Nonlimiting exemplary afucosylated humanized anti-FGFR2IIIb antibodiesalso include antibodies comprising sets of light chain HVR1, HVR2, andHVR3 set forth in SEQ ID NOs: 9, 10, and 11.

In some embodiments, an afucosylated humanized anti-FGFR2IIIb antibodycomprises a heavy chain comprising a variable region sequence that is atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to SEQ ID NO: 4, and wherein the antibody binds FGFR2IIIb. Insome embodiments, an afucosylated humanized anti-FGFR2IIIb antibodycomprises a light chain comprising a variable region sequence that is atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to SEQ ID NO: 5, wherein the antibody binds FGFR2IIIb. In someembodiments, an afucosylated humanized anti-FGFR2IIIb antibody comprisesa heavy chain comprising a variable region sequence that is at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% identicalto SEQ ID NO: 4; and a light chain comprising a variable region sequencethat is at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identical to SEQ ID NO: 5; wherein the antibody binds FGFR2IIIb.

In some embodiments, an afucosylated humanized anti-FGFR2IIIb antibodycomprises at least one of the HVRs discussed herein. That is, in someembodiments, an afucosylated humanized anti-FGFR2IIIb antibody comprisesat least one HVR selected from a heavy chain HVR1 discussed herein, aheavy chain HVR2 discussed herein, a heavy chain HVR3 discussed herein,a light chain HVR1 discussed herein, a light chain HVR2 discussedherein, and a light chain HVR3 discussed herein. Further, in someembodiments, an afucosylated humanized anti-FGFR2IIIb antibody comprisesat least one mutated HVR based on a HVR discussed herein, wherein themutated HVR comprises 1, 2, 3, or 4 amino acid substitutions relative tothe HVR discussed herein. In some embodiments, one or more of the aminoacid substitutions are conservative amino acid substitutions. Oneskilled in the art can select one or more suitable conservative aminoacid substitutions for a particular HVR sequence, wherein the suitableconservative amino acid substitutions are not predicted to significantlyalter the binding properties of the antibody comprising the mutated HVR.

Exemplary afucosylated humanized anti-FGFR2IIIb antibodies also includeantibodies that compete for binding to FGFR2IIIb with an antibody orfragment thereof described herein. Thus, in some embodiments, ahumanized anti-FGFR2IIIb antibody is provided that competes for bindingto FGFR2IIIb with αFGFR2b. In some embodiments, an afucosylatedhumanized anti-FGFR2IIIb antibody is provided that competes for bindingto FGFR2IIIb with αFGFR2b and has at least one activity selected fromenhanced ADCC activity and enhanced affinity for Fc gamma RIIIA (such asFc gamma RIIIA(V158) and/or Fc gamma RIIIA(F158)). In some embodiments,the afucosylated humanized anti-FGFRIIIb antibody does not bind toFGFR2IIIc.

In some embodiments, an afucosylated humanized anti-FGFR2IIIb antibodycomprises one or more human constant regions. In some embodiments, thehuman heavy chain constant region is of an isotype selected from IgA,IgG, and IgD. In some embodiments, the human light chain constant regionis of an isotype selected from κ and λ.

In some embodiments, an afucosylated humanized antibody described hereincomprises a human IgG constant region. In some embodiments, wheneffector function is desirable, an afucosylated humanized anti-FGFR2IIIbantibody comprising a human IgG1 heavy chain constant region or a humanIgG3 heavy chain constant region is selected. In some embodiments, anafucosylated humanized antibody described herein comprises a human IgG1constant region. In some embodiments, an afucosylated humanized antibodydescribed herein comprises a human IgG1 constant region, wherein N297 isnot fucosylated. In some embodiments, an afucosylated human antibodydescribed herein comprises a human IgG1 constant region and a human κlight chain.

In some embodiments, an afucosylated humanized antibody comprises aheavy chain comprising the amino acid sequence of SEQ ID NO: 2 and alight chain comprising the amino acid sequence of SEQ ID NO: 3. In someembodiments, an afucosylated humanized antibody comprises a heavy chainconsisting of the amino acid sequence of SEQ ID NO: 2, and anypost-translational modifications, and a light chain consisting the aminoacid sequence of SEQ ID NO: 3, and any post-translational modifications.

Exemplary Antibody Constant Regions

In some embodiments, an afucosylated antibody described herein comprisesone or more human constant regions. In some embodiments, the human heavychain constant region is of an isotype selected from IgA, IgG, and IgD.In some embodiments, the human light chain constant region is of anisotype selected from κ and λ.

In some embodiments, an afucosylated antibody described herein comprisesa human IgG constant region. In some embodiments, when effector functionis desirable, an afucosylated anti-FGFR2IIIb antibody comprising a humanIgG1 heavy chain constant region or a human IgG3 heavy chain constantregion is selected. In some embodiments, an afucosylated antibodydescribed herein comprises a human IgG1 constant region. In someembodiments, an afucosylated antibody described herein comprises a humanIgG1 constant region, wherein N297 is not fucosylated. In someembodiments, an afucosylated antibody described herein comprises a humanIgG1 constant region and a human κ light chain.

Throughout the present specification and claims unless explicitly statedor known to one skilled in the art, the numbering of the residues in animmunoglobulin heavy chain is that of the EU index as in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991), expresslyincorporated herein by reference. The “EU index as in Kabat” refers tothe residue numbering of the human IgG1 EU antibody.

In certain embodiments, an antibody of the invention comprises a variantFc region has at least one amino acid substitution compared to the Fcregion of a wild-type IgG or a wild-type antibody. In certainembodiments, the variant Fc region has two or more amino acidsubstitutions in the Fc region of the wild-type antibody. In certainembodiments, the variant Fc region has three or more amino acidsubstitutions in the Fc region of the wild-type antibody. In certainembodiments, the variant Fc region has at least one, two or three ormore Fc region amino acid substitutions described herein. In certainembodiments, the variant Fc region herein will possess at least about80% homology with a native sequence Fc region and/or with an Fc regionof a parent antibody. In certain embodiments, the variant Fc regionherein will possess at least about 90% homology with a native sequenceFc region and/or with an Fc region of a parent antibody. In certainembodiments, the variant Fc region herein will possess at least about95% homology with a native sequence Fc region and/or with an Fc regionof a parent antibody.

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibodies with certain improved properties.

In one embodiment, antibodies are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion (i.e., afucosylated antibodies). For example, the amount offucose in a composition comprising a plurality of such antibodies may befrom 0% to about 5%. In some embodiments, a composition comprising aplurality of such antibodies comprises at least 95% afucosylatedantibodies. The amount of fucose is determined by calculating theaverage amount of fucose within the sugar chain at Asn297, relative tothe sum of all glycostructures attached to Asn 297 (e.g., complex,hybrid and high mannose structures). Nonlimiting exemplary methods ofdetecting fucose in an antibody include MALDI-TOF mass spectrometry(see, e.g., WO 2008/077546), HPLC measurement of released fluorescentlylabeled oligosaccharides (see, e.g., Schneider et al., “N-Glycananalysis of monoclonal antibodies and other glycoproteins using UHPLCwith fluorescence detection,” Agilent Technologies, Inc. (2012); Lines,J. Pharm. Biomed. Analysis, 14: 601-608 (1996); Takahasi, J. Chrom.,720: 217-225 (1996)), capillary electrophoresis measurement of releasedfluorescently labeled oligosaccharides (see, e.g., Ma et al., Anal.Chem., 71: 5185-5192 (1999)), and HPLC with pulsed amperometricdetection to measure monosaccharide composition (see, e.g., Hardy, etal., Analytical Biochem., 170: 54-62 (1988)). Asn297 refers to theasparagine residue located at about position 297 in the Fc region (EUnumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. In antibody αFGFR2b described herein, Asn297 is found in thesequence QYNST, and is in bold and underlined in the Table of Sequencesshown below, SEQ ID NO: 2. Fucosylation variants may have improved ADCCfunction. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta,L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples ofpublications related to “afucosylated” or “fucose-deficient” antibodiesinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingafucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US Patent Application No. US 2003/0157108 A1, Presta, L; and WO2004/056312 A1, Adams et al., especially at Example 11), and knockoutcell lines, such as cell lines lacking a functionalalpha-1,6-fucosyltransferase gene, FUT8, e.g., knockout CHO cells (see,e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. etal., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies are further provided with bisected oligosaccharides, e.g., inwhich a biantennary oligosaccharide attached to the Fc region of theantibody is bisected by GlcNAc. Such antibodies may have reducedfucosylation and/or improved ADCC function. Examples of such antibodiesare described, e.g., in WO 2003/011878 (Jean-Mairet et al.); U.S. Pat.No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.).Antibodies with at least one galactose residue in the oligosaccharideattached to the Fc region are also provided. Such antibodies may haveimproved CDC function. Such antibodies are described, e.g., in WO1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764(Raju, S.).

Antibodies are also provided with amino-terminal leader extensions. Forexample, one or more amino acid residues of the amino-terminal leadersequence are present at the amino-terminus of any one or more heavy orlight chains of an antibody. An exemplary amino-terminal leaderextension comprises or consists of three amino acid residues, VHS,present on one or both light chains of an antibody.

The in vivo or serum half-life of human FcRn high affinity bindingpolypeptides can be assayed, e.g., in transgenic mice, in humans, or innon-human primates to which the polypeptides with a variant Fc regionare administered. See also, e.g., Petkova et al. InternationalImmunology 18(12):1759-1769 (2006).

In some embodiments of the invention, an afucosylated antibody mediatesADCC in the presence of human effector cells more effectively than aparent antibody that comprises fucose, Generally, ADCC activity may bedetermined using the in vitro ADCC assay as herein disclosed, but otherassays or methods for determining ADCC activity, e.g. in an animal modeletc., are contemplated.

In certain embodiments, the “K_(D),” “K_(d),” “Kd” or “Kd value” of theantibody is measured by using surface plasmon resonance assays using aBIACORE®-2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) at25° C. with immobilized antigen CM5 chips at ˜10 response units (RU).Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.)are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NETS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, to 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5μL/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1 M ethanolamine isinjected to block unreacted groups. For kinetics measurements, serialdilutions of polypeptide, e.g., full length antibody, are injected inPBS with 0.05% TWEEN-20′ surfactant (PBST) at 25° C. at a flow rate ofapproximately 25 μL/min. Association rates (k_(on)) and dissociationrates (k_(off)) are calculated using a simple one-to-one Langmuirbinding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (Kd) is calculated as the ratiok_(off)/k_(on). See, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999). If the on-rate exceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmonresonance assay above, then the on-rate can be determined by using afluorescent quenching technique that measures the increase or decreasein fluorescence emission intensity (excitation=295 nm; emission=340 nm,16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody in PBS, pH7.2, in the presence of increasing concentrations of antigen as measuredin a spectrometer, such as a stop-flow equipped spectrophometer (AvivInstruments) or a 8000-series SLM-AMINCO™ spectrophotometer(ThermoSpectronic) with a stirred cuvette.

An “on-rate,” “rate of association,” “association rate,” or “k_(on)” ofthe antibody can also be determined as described above using aBIACORE®-2000 or a BIACORE®-3000 system (BIAcore, Inc., Piscataway,N.J.).

In certain embodiments, the difference between said two values (e.g., Kdvalues) is substantially the same, for example, less than about 50%,less than about 40%, less than about 30%, less than about 20%, and/orless than about 10% as a function of the reference/comparator value.

In certain embodiments, the difference between said two values (e.g.,K_(d) values) is substantially different, for example, greater thanabout 10%, greater than about 20%, greater than about 30%, greater thanabout 40%, and/or greater than about 50% as a function of the value forthe reference/comparator molecule.

Exemplary Leader Sequences

In order for some secreted proteins to express and secrete in largequantities, a leader sequence from a heterologous protein may bedesirable. In some embodiments, employing heterologous leader sequencesmay be advantageous in that a resulting mature polypeptide may remainunaltered as the leader sequence is removed in the ER during thesecretion process. The addition of a heterologous leader sequence may berequired to express and secrete some proteins.

Certain exemplary leader sequence sequences are described, e.g., in theonline Leader sequence Database maintained by the Department ofBiochemistry, National University of Singapore. See Choo et al., BMCBioinformatics, 6: 249 (2005); and PCT Publication No. WO 2006/081430.

III. PROPERTIES OF AFUCOSYLATED ANTI-FGFR2IIIB ANTIBODIES

In some embodiments, afucosylated anti-FGFR2IIIb antibodies haveenhanced ADCC activity in vitro and/or in vivo. In some embodiments,afucosylated anti-FGFR2IIIb antibodies have enhanced ADCC activity invitro. In some embodiments, ADCC activity in vitro is determined by amethod described herein, e.g., in Example 3. Briefly,FGFR2IIIb-expressing cells are contacted with freshly isolated humanPBMCs at a ratio of 25:1 effector (PBMCs) to target cells, in thepresence of fucosylated antibody or afucosylated antibody. In someembodiments, Ba/F3 cells that express FGFR2IIIb are used as targetcells. In some embodiments, cytotoxicity is determined by quantifyingLDH release using CytoTox Non-Radioactive Cytotoxicity Assay (Promega,Madison, Wis.). In some embodiments, maximal lysis is determined using5% Triton X-100 and spontaneous release is determined in the absence ofantibody. In some embodiments, the percentage of specific lysis may bedetermined using the formula: (experimental−spontaneousrelease)/(maximal−spontaneous release)×100=% specific lysis. In someembodiments, an afucosylated anti-FGFR2IIIb antibody having enhancedADCC activity results in specific lysis that is at least 10, at least15, at least 20, at least 25, at least 30, at least 35, at least 40, atleast 45, at least 50, at least 60, at least 65, at least 70, or atleast 75 percentage points greater than specific lysis with the sameamount of a fucosylated antibody, at at least one concentration ofantibody tested. In some embodiments, an afucosylated anti-FGFR2IIIbantibody having enhanced ADCC activity results in specific lysis that isat least 10, at least 15, at least 20, at least 25, at least 30, atleast 35, at least 40, at least 45, at least 50, at least 60, at least65, at least 70, or at least 75 percentage points greater than specificlysis with a fucosylated antibody, where each antibody is at aconcentration of between 0.01 and 1 μg/ml and the target cells are Ba/F3cells expressing FGFR2IIIb. In some embodiments, the antibodies aretested at a concentration of 0.01 μg/ml, 0.1 μg/ml, or 1 μg/ml.

In some embodiments, afucosylated anti-FGFR2IIIb antibodies haveenhanced affinity for Fc gamma RIIIA In some embodiments, afucosylatedanti-FGFR2IIIb antibodies have enhanced affinity for Fc gammaRIIIA(V158). In some embodiments, afucosylated anti-FGFR2IIIb antibodieshave enhanced affinity for Fc gamma RIIIA(F158). In some embodiments,antibody affinity for Fc gamma RIIIA is determined using surface plasmonresonance, e.g., as described herein in Example 2, and/or as follows,which is described with reference to Fc gamma RIIIA(V158), but which isalso suitable for determining affinity for Fc gamma RIIIA(F158).Briefly, in some embodiments, fucosylated or afucosylated anti-FGFR2IIIbantibody is captured on a protein A-coated dextran chip. Fc gamma RIIIA(V158) (available from, e.g., R&D Systems) is injected at variousconcentrations. The association constant, dissociation constant, andaffinity of Fc gamma RIIIA (V158) for fucosylated and afucosylatedanti-FGFR2IIIb antibody may be determined, e.g., using software providedwith the surface plasmon resonance system (for example, Biacore T200Evaluation Software 1:1 binding model). In some embodiments, anafucosylated anti-FGFR2IIIb antibody with enhanced affinity for Fc gammaRIIIA (such as Fc gamma RIIIA(V158) or Fc gamma RIIIA(F158)) binds to Fcgamma RIIIA with at least 2-fold, at least 3-fold, at least 4-fold, atleast 5-fold, at least 7-fold, at least 10-fold, at least 12-fold, atleast 15-fold, at least 17-fold, or at least 20-fold greater affinitythan a fucosylated anti-FGFR2IIIb antibody. For example, if anafucosylated anti-FGFR2IIIb antibody binds to Fc gamma RIIIA (V158) withan affinity (K_(D)) of 9.2 nM and a fucosylated anti-FGFR2IIIb antibodybinds to Fc gamma RIIIA (V158) with an affinity (K_(D)) of 207 nM, thenthe afucosylated anti-FGFR2IIIb antibody binds to Fc gamma RIIIA (V158)with 207/9.2=22.5-fold greater affinity than the fucosylatedanti-FGFR2IIIb antibody.

IV. ANTI-FGFR2IIIB ANTIBODY EXPRESSION AND PRODUCTION Nucleic AcidMolecules Encoding Anti-FGFR2IIIb Antibodies

Nucleic acid molecules comprising polynucleotides that encode one ormore chains of anti-FGFR2IIIb antibodies are provided. In someembodiments, a nucleic acid molecule comprises a polynucleotide thatencodes a heavy chain or a light chain of an anti-FGFR2IIIb antibody. Insome embodiments, a nucleic acid molecule comprises both apolynucleotide that encodes a heavy chain and a polynucleotide thatencodes a light chain, of an anti-FGFR2IIIb antibody. In someembodiments, a first nucleic acid molecule comprises a firstpolynucleotide that encodes a heavy chain and a second nucleic acidmolecule comprises a second polynucleotide that encodes a light chain.

In some such embodiments, the heavy chain and the light chain areexpressed from one nucleic acid molecule, or from two separate nucleicacid molecules, as two separate polypeptides. In some embodiments, suchas when an antibody is an scFv, a single polynucleotide encodes a singlepolypeptide comprising both a heavy chain and a light chain linkedtogether.

In some embodiments, a polynucleotide encoding a heavy chain or lightchain of an anti-FGFR2IIIb antibody comprises a nucleotide sequence thatencodes a leader sequence, which, when translated, is located at the Nterminus of the heavy chain or light chain. As discussed above, theleader sequence may be the native heavy or light chain leader sequence,or may be another heterologous leader sequence.

Nucleic acid molecules may be constructed using recombinant DNAtechniques conventional in the art. In some embodiments, a nucleic acidmolecule is an expression vector that is suitable for expression in aselected host cell.

Vectors

Vectors comprising polynucleotides that encode anti-FGFR2IIIb heavychains and/or anti-FGFR2IIIb light chains are provided. Vectorscomprising polynucleotides that encode anti-FGFR2IIIb heavy chainsand/or anti-FGFR2IIIb light chains are also provided. Such vectorsinclude, but are not limited to, DNA vectors, phage vectors, viralvectors, retroviral vectors, etc. In some embodiments, a vectorcomprises a first polynucleotide sequence encoding a heavy chain and asecond polynucleotide sequence encoding a light chain. In someembodiments, the heavy chain and light chain are expressed from thevector as two separate polypeptides. In some embodiments, the heavychain and light chain are expressed as part of a single polypeptide,such as, for example, when the antibody is an scFv.

In some embodiments, a first vector comprises a polynucleotide thatencodes a heavy chain and a second vector comprises a polynucleotidethat encodes a light chain. In some embodiments, the first vector andsecond vector are transfected into host cells in similar amounts (suchas similar molar amounts or similar mass amounts). In some embodiments,a mole- or mass-ratio of between 5:1 and 1:5 of the first vector and thesecond vector is transfected into host cells. In some embodiments, amass ratio of between 1:1 and 1:5 for the vector encoding the heavychain and the vector encoding the light chain is used. In someembodiments, a mass ratio of 1:2 for the vector encoding the heavy chainand the vector encoding the light chain is used.

In some embodiments, a vector is selected that is optimized forexpression of polypeptides in CHO or CHO-derived cells, or in NSO cells.Exemplary such vectors are described, e.g., in Running Deer et al.,Biotechnol. Prog. 20:880-889 (2004).

Host Cells

In various embodiments, anti-FGFR2IIIb heavy chains and/oranti-FGFR2IIIb light chains may be expressed in prokaryotic cells, suchas bacterial cells; or in eukaryotic cells, such as fungal cells (suchas yeast), plant cells, insect cells, and mammalian cells. Suchexpression may be carried out, for example, according to proceduresknown in the art. Exemplary eukaryotic cells that may be used to expresspolypeptides include, but are not limited to, COS cells, including COS 7cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S,DG44. Lec13 CHO cells, and FUT8 CHO cells; PER.C6® cells (Crucell); andNSO cells. In some embodiments, anti-FGFR2IIIb heavy chains and/oranti-FGFR2IIIb light chains may be expressed in yeast. See, e.g., U.S.Publication No. US 2006/0270045 A1. In some embodiments, a particulareukaryotic host cell is selected based on its ability to make desiredpost-translational modifications to the anti-FGFR2IIIb heavy chainsand/or anti-FGFR2IIIb light chains. For example, in some embodiments,CHO cells produce polypeptides that have a higher level of sialylationthan the same polypeptide produced in 293 cells.

Introduction of one or more nucleic acids into a desired host cell maybe accomplished by any method, including but not limited to, calciumphosphate transfection, DEAE-dextran mediated transfection, cationiclipid-mediated transfection, electroporation, transduction, infection,etc. Nonlimiting exemplary methods are described, e.g., in Sambrook etal., Molecular Cloning, A Laboratory Manual, 3^(rd) ed. Cold SpringHarbor Laboratory Press (2001). Nucleic acids may be transiently orstably transfected in the desired host cells, according to any suitablemethod.

In some embodiments afucosylated anti-FGFR2IIIb antibodies are producedin cells capable of producing afucosylated antibodies, such as Lec13 CHOcells deficient in protein fucosylation (Ripka et al. Arch. Biochem.Biophys. 249:533-545 (1986); US Patent Application No. US 2003/0157108A1, Presta, L; and WO 2004/056312 A1, Adams et al., especially atExample 11), and knockout cell lines, such as cell lines lacking afunctional alpha-1,6-fucosyltransferase gene, FUT8, e.g., knockout CHOcells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004);Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); andWO2003/085107). In some embodiments, afucosylated anti-FGFR2IIIbantibodies are produced in CHO cells lacking a functional FUT8 gene. Insome embodiments, afucosylated anti-FGFR2IIIb antibodies are produced inPotelligent® CHOK1SV cells (BioWa/Lonza, Allendale, N.J.).

Purification of Anti-FGFR2IIIb Antibodies

Anti-FGFR2IIIb antibodies may be purified by any suitable method. Suchmethods include, but are not limited to, the use of affinity matrices orhydrophobic interaction chromatography. Suitable affinity ligandsinclude the FGFR2IIIb ECD and ligands that bind antibody constantregions. For example, a Protein A, Protein G, Protein A/G, or anantibody affinity column may be used to bind the constant region and topurify an anti-FGFR2IIIb antibody. Hydrophobic interactivechromatography, for example, a butyl or phenyl column, may also suitablefor purifying some polypeptides. Many methods of purifying polypeptidesare known in the art.

Cell-Free Production of Anti-FGFR2IIIb Antibodies

In some embodiments, an anti-FGFR2IIIb antibody is produced in acell-free system. Nonlimiting exemplary cell-free systems are described,e.g., in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009);Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al., Biotechnol.Adv. 21: 695-713 (2003).

V. THERAPEUTIC COMPOSITIONS AND METHODS Methods of Treating DiseasesUsing Anti-FGFR2IIIb Antibodies

Antibodies of the invention, and compositions comprising antibodies ofthe invention, are provided for use in methods of treatment for humansor animals. Methods of treating disease comprising administeringafucosylated anti-FGFR2IIIb antibodies are also provided. Nonlimitingexemplary diseases that can be treated with afucosylated anti-FGFR2IIIbantibodies include, but are not limited to cancer. In some embodiments,methods of treating cancer are provided, comprising administering anafucosylated anti-FGFR2IIIb antibody. In some embodiments, the cancer isselected from gastric cancer, breast cancer, ovarian cancer, endometrialcancer, pancreatic cancer, or esophageal cancer. In some embodiments,methods of treating gastric cancer are provided, comprisingadministering an afucosylated anti-FGFR2IIIb antibody.

In some embodiments, a cancer comprises an FGFR2 gene amplification. Insome embodiments, a cancer comprising an FGFR2 gene amplificationoverexpresses FGFR2IIIb. In some embodiments, a cancer comprising FGFR2amplification overexpresses FGFR2IIIb to a greater extent thanFGFR2IIIc. In some embodiments, a cancer comprising FGFR2 amplificationexpresses FGFR2IIIb at a normalized level that is more than 2-fold,3-fold, 5-fold, or 10-fold greater than the normalized level ofFGFR2IIIc expression. In some embodiments, the expression levels arenormalized to GUSB. In some embodiments, a cancer overexpressesFGFR2IIIb but does not comprise FGFR2 gene amplification. In someembodiments, a gastric cancer comprises an FGFR2 gene amplification. Insome embodiments, a gastric cancer comprising an FGFR2 geneamplification overexpresses FGFR2IIIb. In some embodiments, a gastriccancer comprising FGFR2 amplification overexpresses FGFR2IIIb to agreater extent than FGFR2IIIc. In some embodiments, a gastric cancercomprising FGFR2 amplification expresses FGFR2IIIb at a normalized levelthat is more than 2-fold, 3-fold, 5-fold, or 10-fold greater than thenormalized level of FGFR2IIIc expression. In some embodiments, theexpression levels are normalized to GUSB. In some embodiments, a gastriccancer overexpresses FGFR2IIIb but does not comprise FGFR2 geneamplification. In some embodiments, overexpression is mRNAoverexpression. In some embodiments, overexpression is proteinoverexpression.

FGFR2IIIb gene amplification may be determined by any suitable method inthe art, including but not limited to, in situ hybridization (ISH). Insome embodiments, FGFR2 amplification comprises FGFR2:CEN10 (chromosome10 centromere) ratio of >3.

FGFR2IIIb mRNA overexpression may be determined by any suitable methodin the art, including but not limited to, methods comprisingquantitative PCR (qPCR). The term “FGFR2IIIb mRNA overexpression” meanselevated levels of FGFR2IIIb mRNA, regardless of the cause of suchelevated levels (i.e., whether the elevated levels are a result ofincreased transcription and/or decreased degradation of mRNA, othermechanism, or a combination of mechanisms).

FGFR2IIIb protein overexpression may be determined by any suitablemethod in the art, including but not limited to, antibody-based methodssuch as immunohistochemistry (IHC). In some embodiments, the IHCstaining is scored according to methods in the art. The term “FGFR2IIIbprotein overexpression” means elevated levels of FGFR2IIIb protein,regardless of the cause of such elevated levels (i.e., whether theelevated levels are a result of increased translation and/or decreaseddegradation of protein, other mechanism, or a combination ofmechanisms). In some embodiments, 1+, 2+, or 3+ staining of tumor cellsby IHC indicates FGFR2IIIb overexpression. In some embodiments, 2+ or 3+staining of tumor cells by IHC indicates FGFR2IIIb overexpression. Insome embodiments, the IHC staining is scored as described in Example 6.

Pharmaceutical Compositions

In various embodiments, compositions comprising afucosylatedanti-FGFR2IIIb antibodies are provided in formulations with a widevariety of pharmaceutically acceptable carriers (see, e.g., Gennaro,Remington: The Science and Practice of Pharmacy with Facts andComparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al.,Pharmaceutical Dosage Forms and Drug Delivery Systems, 7^(th) ed.,Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook ofPharmaceutical Excipients, 3^(rd) ed., Pharmaceutical Press (2000)).Various pharmaceutically acceptable carriers, which include vehicles,adjuvants, and diluents, are available. Moreover, variouspharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are also available. Non-limiting exemplary carriersinclude saline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof.

In various embodiments, compositions comprising afucosylatedanti-FGFR2IIIb antibodies may be formulated for injection, includingsubcutaneous administration, by dissolving, suspending, or emulsifyingthem in an aqueous or nonaqueous solvent, such as vegetable or otheroils, synthetic aliphatic acid glycerides, esters of higher aliphaticacids, or propylene glycol; and if desired, with conventional additivessuch as solubilizers, isotonic agents, suspending agents, emulsifyingagents, stabilizers and preservatives. In various embodiments, thecompositions may be formulated for inhalation, for example, usingpressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen, and the like. The compositions may also beformulated, in various embodiments, into sustained releasemicrocapsules, such as with biodegradable or non-biodegradable polymers.A non-limiting exemplary biodegradable formulation includes poly lacticacid-glycolic acid polymer. A non-limiting exemplary non-biodegradableformulation includes a polyglycerin fatty acid ester. Certain methods ofmaking such formulations are described, for example, in EP 1 125 584 A1.

Routes of Administration

In various embodiments, afucosylated anti-FGFR2IIIb antibodies may beadministered in vivo by various routes, including, but not limited to,oral, intra-arterial, parenteral, intranasal, intramuscular,intracardiac, intraventricular, intratracheal, buccal, rectal,intraperitoneal, intradermal, topical, transdermal, and intrathecal, orotherwise by implantation or inhalation. The subject compositions may beformulated into preparations in solid, semi-solid, liquid, or gaseousforms; including, but not limited to, tablets, capsules, powders,granules, ointments, solutions, suppositories, enemas, injections,inhalants, and aerosols. The appropriate formulation and route ofadministration may be selected according to the intended application.

Pharmaceutical compositions are administered in an amount effective fortreatment or prophylaxis of cancer, such as gastric cancer, breastcancer, ovarian cancer, endometrial cancer, pancreatic cancer, oresophageal cancer. The therapeutically effective amount is typicallydependent on the weight of the subject being treated, his or herphysical or health condition, the extensiveness of the condition to betreated, or the age of the subject being treated. In general,afucosylated anti-FGFR2IIIb antibodies may be administered in an amountin the range of about 10 μg/kg body weight to about 100 mg/kg bodyweight per dose. In some embodiments, afucosylated anti-FGFR2IIIbantibodies may be administered in an amount in the range of about 50μg/kg body weight to about 5 mg/kg body weight per dose. In someembodiments, anti-FGFR2IIIb antibodies may be administered in an amountin the range of about 100 μg/kg body weight to about 10 mg/kg bodyweight per dose. In some embodiments, afucosylated anti-FGFR2IIIbantibodies may be administered in an amount in the range of about 100μg/kg body weight to about 20 mg/kg body weight per dose. In someembodiments, afucosylated anti-FGFR2IIIb antibodies may be administeredin an amount in the range of about 0.5 mg/kg body weight to about 20mg/kg body weight per dose.

The afucosylated anti-FGFR2IIIb antibody compositions may beadministered as needed to subjects. Determination of the frequency ofadministration may be made by persons skilled in the art, such as anattending physician based on considerations of the condition beingtreated, age of the subject being treated, severity of the conditionbeing treated, general state of health of the subject being treated andthe like. In some embodiments, an effective dose of an afucosylatedanti-FGFR2IIIb antibody is administered to a subject one or more times.In various embodiments, an effective dose of an afucosylatedanti-FGFR2IIIb antibody is administered to the subject once a month,more than once a month, such as, for example, every two months or everythree months. In other embodiments, an effective dose of an afucosylatedanti-FGFR2IIIb antibody is administered less than once a month, such as,for example, every two weeks or every week. An effective dose of anafucosylated anti-FGFR2IIIb antibody is administered to the subject atleast once. In some embodiments, the effective dose of an afucosylatedanti-FGFR2IIIb antibody may be administered multiple times, includingfor periods of at least a month, at least six months, or at least ayear.

Combination Therapy

Afucosylated anti-FGFR2IIIb antibodies may be administered alone or withother modes of treatment. They may be provided before, substantiallycontemporaneous with, or after other modes of treatment, for example,surgery, chemotherapy, or radiation therapy. In some embodiments, anafucosylated anti-FGFR2IIIb antibody is administered in conjunction withanother anti-cancer agent. Nonlimiting exemplary anti-cancer agents thatmay be administered with an afucosylated anti-FGFR2IIIb antibody includeplatinum agents (such as cisplatin, oxaliplatin, and carboplatin),paclitaxel (TAXOL®), albumin-engineered nanoparticle formulation ofpaclitaxel (ABRAXANE®), docetaxel (TAXOTERE®), gemcitabine (GEMZAR®),capecitabine (XELODA®), irinotecan (CAMPTOSAR®), epirubicin (ELLENCE®,PHARMORUBICIN®), FOLFOX (oxaliplatin combined with 5-FU and leucovorin),FOLFIRI (combination of leucovorin, 5-FU and irinotecan), leucovorin,fluorouracil (5-FU, EFUDEX®), mitomycin C (MITOZYTREX™, MUTAMYCIN®), anddoxorubicin hydrochloride (Adriamycin PFS, Adriamycin RDF, RUBEX®). Insome embodiments, an afucosylated anti-FGFR2IIIb antibody isadministered in conjunction with paclitaxel. In some embodiments, anafucosylated anti-FGFR2IIIb antibody is administered in conjunction withcisplatin and/or 5-FU. In some embodiments, an afucosylatedanti-FGFR2IIIb antibody is administered in conjunction with cisplatinand 5-FU. In some embodiments, an afucosylated anti-FGFR2IIIb antibodyis administered in conjunction with FOLFOX (oxaliplatin, 5-FU, andleucovorin).

Kits/Articles of Manufacture

The invention also provides kits, medicines, compositions, and unitdosage forms for use in any of the methods described herein.

Kits of the invention include one or more containers comprising anafucosylated anti-FGFR2IIIb antibody (or unit dosage forms and/orarticles of manufacture). In some embodiments, a unit dosage is providedwherein the unit dosage contains a predetermined amount of a compositioncomprising an afucosylated anti-FGFR2IIIb antibody, with or without oneor more additional agents. In some embodiments, such a unit dosage issupplied in single-use prefilled syringe for injection. In variousembodiments, the composition contained in the unit dosage may comprisesaline, sucrose, or the like; a buffer, such as phosphate, or the like;and/or be formulated within a stable and effective pH range.Alternatively, in some embodiments, the composition may be provided as alyophilized powder that may be reconstituted upon addition of anappropriate liquid, for example, sterile water. In some embodiments, thecomposition comprises one or more substances that inhibit proteinaggregation, including, but not limited to, sucrose and arginine. Insome embodiments, a composition of the invention comprises heparinand/or a proteoglycan.

In some embodiments, kits of the invention further comprise instructionsfor use in the treatment of, e.g., cancer (such as gastric cancer,breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer, oresophageal cancer) in accordance with any of the methods describedherein. The kit may further comprise a description of selection anindividual suitable or treatment. Instructions supplied in the kits ofthe invention are typically written instructions on a label or packageinsert (e.g., a paper sheet included in the kit), but machine-readableinstructions (e.g., instructions carried on a magnetic or opticalstorage disk) are also acceptable. In some embodiments, the kit furthercomprises another therapeutic agent.

The kits of the invention are in suitable packaging. Suitable packaginginclude, but is not limited to, vials, bottles, jars, flexible packaging(e.g., sealed Mylar or plastic bags), and the like. Kits may optionallyprovide additional components such as buffers and interpretativeinformation. The present application thus also provides articles ofmanufacture, which include vials (such as sealed vials), bottles, jars,flexible packaging, and the like.

EXAMPLES

The examples discussed below are intended to be purely exemplary of theinvention and should not be considered to limit the invention in anyway. The examples are not intended to represent that the experimentsbelow are all or the only experiments performed. Efforts have been madeto ensure accuracy with respect to numbers used (for example, amounts,temperature, etc.) but some experimental errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is weight average molecular weight, temperature is indegrees Centigrade, and pressure is at or near atmospheric.

Example 1 Production of Afucosylated Anti-FGFR2IIIb Antibody

Construction of Expression Vector.

Nucleotide sequences encoding the heavy chain (HC) and light chain (LC)of monoclonal antibody αFGFR2b were cloned into the GS Gene ExpressionSystem (Lonza, Basel, Switzerland) according to the manufacturer'sinstructions. The system generates a double gene vector (DGV) containingthe expression cassettes for both the light chain and the heavy chain.

Choice of Host Cell Line.

To generate afucosylated monoclonal antibody αFGFR2bA (the designation“A” after αFGFR2b refers to “afucosylated”), the Potelligent® CHOK1SVcells (BioWa/Lonza, Allendale, N.J.) was chosen as host cell line.Potelligent® CHOK1SV cells lack the FUT8 gene (α1,6-fucosyltransferase)and therefore produce fucose-free antibodies (afucosylated antibodies).

Construction of Stable Cell Line for Production of Afucosylated αFGR2bAAntibody:

The expression vector comprising the nucleotide sequences encodingαFGFR2b antibody heavy chain and light chain described above transfectedinto Potelligent® CHOK1SV cells by electroporation according to themanufacturer's instructions. Electroporated cells were seeded into a96-well plate at about 10,000 cells/50 μl/well in CD CHO medium withoutL-glutamine. Selective CD CHO medium containing 67 μM L-methioninesulfoximine (MSX, SIGMA cat #M5379, St. Louis, Mo.) was added the nextday at 150 Cell growth and clone formation was monitored with an IN CellAnalyzer 2000 (GE Healthcare, Piscataway, N.J.).

After 4-6 weeks, the surviving colonies were screened for expression ofαFGFR2bA antibody using a Homogenous Time Resolved Fluorescence (HTRF)based assay against standard curves generated with purified αFGFR2bantibody, using XL665 conjugated protein A and cryptate conjugatedpolyclonal rabbit IgG (Cisbio, Bedford, Mass.). The highest expressingclones were expanded through a series of increasing-scale productionprocesses, including 24-well plates, spin tubes, shake flasks, benchscale bioreactors, and finally a platform production process. At eachstep, only a subset comprising the highest-expressing clones were takenon to the next process. The final production clone was selected based onthe evaluation of protein product titers, cell growth characteristics,product quality, stability, as well as scalability in bioreactors. Thefinal production line had an expression level of about 3.5 g/L forαFGFR2bA. Lack of fucosylation in αFGFR2bA produced in the finalproduction cell line was confirmed using normal phase HLPC (N-HPLC)chromatography.

αFGFR2bA antibody was purified by column chromatography andultrafiltration to concentrate the purified material, then diafiltrationto exchange into formulation buffer (20 mM histidine, 150 mM L-arginine,0.01% polysorbate 20, pH 6.0). Antibody was stored below −70° C.

Glycan analysis of αFGFR2bA is performed by releasing the glycans fromthe protein, labeling the glycans with anthranilic acid (2-AA), and thenpurifying the labeled glycans. Normal phase HPLC with fluorescentdetection is used to separate the glycans and measure the relativeamount of each glycan in the antibody. FIG. 7 shows that αFGFR2b derivedfrom the Potelligent® CHOK1SV cell line and αFGFR2b produced fromCHOK1SV had two different glycan distributions. (A) Glycan distributionof αFGFR2b derived from the Potelligent® CHOK1SV cell line shows thatthe antibody lacks fucose (“G0”). (B) Glycan distribution of αFGFR2bderived from the CHOK1SV cell line shows that the antibody containsfucose (“G0F”).

Glycan peaks from the normal phase HPLC separation were identified usingtwo orthogonal methods. First, the Potelligent® CHOK1SV produced αFGFR2bwere labeled and separated using the normal phase HPLC method. After thefluorescent detection, the glycans were passed through a QTrap massspectrometer. The mass of each peak was determined and used topositively identify each glycan, and is shown in Table 2.

TABLE 2 Mass of glycan peaks Observed Theoretical Mass Glycan Mass (Da)(Da) G0 1437 1437.4 Man-5 1355 1355.3 G1 1600 1599.4

The mass of each fucosylated form of the glycans (G0F, G1F, and G2F)were also searched in the Potelligent® CHOK1SV produced αFGFR2b and werenot observed. FIG. 8 shows schematic diagrams of the G0, G1, G2, G0F,G1F, G2F, and mannose-5 (or Man-5) glycan structures typically observedin antibodies.

The peak identities from the HPLC assay were also confirmed using glycanstandards purchased from Prozyme and matching the retention time betweenthe standards and the αFGFR2 profiles from both the Potelligent® CHOK1SVcell line and the CHOK1SV cell line. These standards were able toidentify G0, G0F, Man5, G1, G1F, and G2F.

The results from the HPLC assay as well as the characterization dataconfirmed the lack of fucosylation in αFGFR2 derived from thePotelligent® CHOK1SV cell line.

Example 2 Afucosylated Anti-FGFR2b Antibody Binding Affinity

The binding affinities of αFGFR2bA and αFGFR2bF (the designation “F”after αFGFR2b refers to “fucosylated”) for Fc gamma RIIIA(V158) weredetermined by surface plasmon resonance. Briefly, Protein A wascovalently attached to a dextran chip using the Amine Coupling Kit (GEHealthcare Life Sciences, Piscataway, N.J.) and 100 mM ethylenediamine(Sigma) in 100 mM sodium borate buffer, pH 8.0 (Sigma, St. Louis, Mo.)as the blocking reagent. Approximately 600-800 RU of αFGFR2bA andαFGFR2bF were captured on separate flow cells and a Protein A (Pierce)derivatized flow cell served as a reference control. Fc gamma RIIIA(V158) (R&D Systems, Minneapolis, Minn.) was diluted in HBS-P+ runningbuffer (Biacore, GE Healthcare, Piscataway, N.J.) and injected at 5concentrations (0 nM, 12.3 nM, 37 nM, 111 nM, 333 nM, and 1000 nM) induplicate. The association constant, dissociation constant, and affinityfor αFGFR2bA were calculated using the Biacore T200 Evaluation Software1:1 binding model. The affinity constant for αFGFR2bF binding wasdetermined using the Biacore T200 Evaluation Software steady stateaffinity model. The results are shown in Table 3.

TABLE 3 αFGFR2bA and αFGFR2bF antibody affinities for Fc gammaRIIIA(V158) k_(a) K_(D) (k_(d)/k_(a)) (1/Ms) × k_(d) αFGFR2b (nM)10{circumflex over ( )}3 (1/ms) A 9.2 940.1 8.6 F 207 — —

As shown in Table 3, Fc gamma RIIIA(V158) bound αFGFR2bA with more than20-fold greater affinity than it bound αFGFR2bF.

Example 3 Afucosylated Anti-FGFR2b Antibody ADCC Activity

In vitro assays to determine the ADCC activity of αFGFR2bA antibodyversus αFGFR2bF antibody were performed. Ba/F3 FGFR2IIIb-expressingtarget cells were produced as follows. Ba/F3 cells acquired fromLeibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ, cat # ACC300, Braunschweig, Germany) weremaintained in RPMI (Mediatech, cat #10-041-CV, Manassas, Va.),supplemented with 10% fetal bovine serum (Mediatech, cat #35-010-CV), 1ng/mL murine IL-3 (Peprotech, cat #213-13, Rocky Hill, N.J.), 1×BME(Invitrogen, cat #1047574, Grand Island, N.Y.), and1×Penicillin-streptomycin (Mediatech cat #30-002-C1). The Ba/F3 cellswere transfected with an expression vector that expresses FGFR2IIIb,pBNew-hFGFR2b, using Cell Line Nucleofector® Kit V (Lonza, cat #VCA-1003) following the manufacturer's protocol. pBNew vector contains aCAG promoter with a chicken β-actin intron and ampicillin and puromycingrowth selection genes. Transfected cells were incubated in full growthmedia for 3 days then treated with 2 μg/mL of puromycin (InVivoGen, cat# ant-pr-1, San Diego, Calif.). Puromycin selection was maintainedthroughout culturing. To generate individual stable clones, cells wereplated at a density of 1 cell per 3 wells. Fluorescent activated cellsorting (FACS) with an anti-FGFR2IIIb antibody was used to select theclones with the highest level of FGFR2IIIb expression.

MFM-223 cells were obtained from the Health Protection Agency, UK;OCUM-2M cells were obtained from Osaka City University, Osaka, Japan;and KATO III cells were obtained from ATCC, Rockville, Md. All cellswere cultured using standard methods. Freshly isolated PMBC from healthydonors were obtained from AllCells, Emeryville, Calif.

ADCC assays were conducted using effector cells from 3 independentdonors on 3 different days. ADCC assay testing was performed usingfreshly isolated human PBMCs as effector cells at an effector to target(E/T) cell ratio of 25:1. The target cells were incubated for 16 hoursin the presence of effectors and increasing concentrations of antibody.The ADCC assay was validated using 2 positive control antibodies,HERCEPTIN® on SKOV-3 target cells and RITUXIN® on Raji target cells. Afully human IgG1 negative control antibody (Eureka Therapeutics, CatalogET901, Emeryville, Calif.) was used for non-specific cell cytotoxicity.Cytotoxicity was determined by quantifying LDH release as per themanufacturer's instructions (CytoTox Non Radioactive Cytotoxicity Assay,Promega, Madison, Wis.).

Maximal lysis was determined in the presence of 5% Triton X-100, andspontaneous release was determined in the absence of antibody.Percentage of specific lysis was calculated as follows, as a percentageof maximal lysis less spontaneous release: (experimental−spontaneousrelease)/(maximal−spontaneous release)×100=% specific lysis

The results for the Ba/F3 cells are shown in FIG. 1. AfucosylatedαFGFR2bA antibody induced greater specific lysis than fucosylatedαFGFR2bF antibody in FGFR2IIIb-expressing Ba/F3 cells. In OCUM-2M, MFM223, and KATO III cells, αFGFR2bA showed greater ADCC activity at lowerconcentrations than αFGFR2bF, although maximal specific lysis of theantibodies was comparable in OCUM-2M and MFM 223 cells (data not shown).Further, αFGFR2b showed little to no ADCC activity inFGFR2IIIc-expressing Ba/F3 cells. See FIG. 1A.

Example 4 Afucosylated Anti-FGFR2b Antibody Activity in Gastric Cancerand Breast Cancer Xenograft Models

Six week old female CB 17 SCID mice were purchased from Charles RiverLaboratories (Wilmington, Mass.) and were acclimated for 1 week beforethe start of the study. Human gastric carcinoma cell line OCUM-2M orbreast carcinoma cell line MFM-223 were used as the tumor models.OCUM-2M was purchased from Public University Corporation Osaka CityUniversity (OCU, Osaka, Japan), and MFM-223 was purchased from CultureCollections, Public Health England (98050130, HPA Culture Collections,Salisbury, UK). The cells were cultured for up to three passages incomplete growth medium to expand for implantation. OCUM-2M and MFM-223cells were cultured in Dulbecco's Minimum Essential Medium (DMEM) andMinimum Essential Medium (MEM), respectively. All medium wassupplemented with 10% heat-inactivated Fetal Bovine Serum (FBS), 2 mML-Glutamine, and Penicillin-Streptomycin solution. Cells were grown at37° C. in a humidified atmosphere with 5% CO₂.

When the cultured cells reached 85-90% confluence, cells were harvestedand resuspended in cold Ca²⁺ and Mg²⁺ free phosphate buffered saline(PBS) containing 50% Matrigel (BD BioSciences, San Jose, Calif.).OCUM-2M cells were implanted subcutaneously over the right flank of themice at 5×10⁵ cells/100 μl/mouse. MFM-223 cells were implantedsubcutaneously over the right flank of the mice at 5×10⁵ cells/100μl/mouse, and 0.72 mg 90-day release 17-β estradiol pellets (InnovativeResearch of America, Sarasota, Fla.) were inoculated subcutaneously intothe right flank. Mice were monitored twice weekly following cellimplantation for tumor growth. Tumor size was measured according to theformula: Tumor size (mm³)=(width (mm)×length (mm)²)/2. When MFM-223tumors reached 80 mm³ mice were sorted and randomized (n=10), andtreatment was initiated. For OCUM-2M tumors, single therapy withanti-FGFR2b was initiated once tumors reached an average size of 100mm³, and combination therapy was initiated once tumors reached anaverage size of 250 mm³.

Anti-FGFR2IIIb humanized antibody (Afucosylated, αFGFR2bA; Fucosylated,αFGFR2bF) or albumin as a negative control was administered at dosesranging from 1 to 10 mg/kg via intraperitoneal injection twice per weekas specified in the figure legends. Chemotherapeutic agents wereadministered via intraperitoneal injection twice per week at 12 mg/kgfor paclitaxel, 2.3 mg/kg for fluorouracil (5-FU), and 33 mg/kg forcisplatin. Upon initiation of therapy, tumor sizes were measured in eachmouse twice weekly. The length and width of each tumor was measuredusing calipers and the tumor size calculated according to the formulaabove. Mice were euthanized when the subcutaneous tumor volumes exceeded2000 mm³ or when the tumors became excessively necrotic.

Comparisons of tumor volume as a consequence of were determined to bestatistically significant if P<0.05. P-values were calculated usingunpaired, two-tailed t-test analyses of the calculated tumor volumes onthe final day upon which tumors were measured.

FIG. 2 shows efficacy of αFGFR2bA and αFGFR2bF at (A and B) 10 mg/kg and(C and D) 3 mg/kg in an OCUM-2M human gastric cancer xenograft modelwith FGFR2 amplification. (A) At 10 mg/kg, both fucosylated andafucosylated antibody induced immediate tumor regression. However,afucosylated αFGFR2bA induced a more durable response than fucosylatedαFGFR2bF. (B) Afucosylated αFGFR2bA significantly reduced final OCUM-2Mtumor size compared to fucosylated αFGFR2bF (p=0.0153). Statisticalsignificance was determined via two-tailed, unpaired t-Test. (C) Whendosed at 3 mg/kg, afucosylated αFGFR2bA reduced tumor growth compared tofucosylated αFGFR2bF. Indeed, afucosylated αFGFR2bA induced greatertumor regression and durable response compared to fucosylated αFGFR2bF(note: one animal was removed from the αFGFR2bF group at about day 42,resulting in a shift in the curve). (D) Afucosylated αFGFR2bA at 3 mg/kgcompared to fucosylated αFGFR2bF notably reduced final OCUM-2M tumorsize (p=0.0767). Statistical significance was determined via two-tailed,unpaired t-Test.

FIG. 3 shows dose-dependent tumor inhibition by αFGFR2bA. (A) SCID micebearing subcutaneous OCUM-2M xenografts were treated with 1, 1.5, 2, 3,or 5 mg/kg afucosylated αFGFR2bA when average tumor size reachedapproximately 100 mm³. Although all doses of afucosylated αFGFR2bAinhibited tumor growth, the greatest suppression and durable responsewere observed with 3 and 5 mg/kg, with reduced growth suppressionobserved with 2, 1.5, and 1 mg/kg αFGFR2bA. (B) Afucosylated αFGFR2bAreduced final OCUM-2M tumor size, with higher doses showing more potentgrowth suppression. Tumor growth suppression was statisticallysignificant for all doses (5 mg/kg, p<0.0001; 3 mg/kg, p<0.0001; 2mg/kg, p<0.0001; 1.5 mg/kg, p=0.0003; 1 mg/kg, p=0.0013). Statisticalsignificance was determined via two-tailed, unpaired t-Test.

FIG. 4 shows enhancement of paclitaxel anti-tumor activity in an OCUM-2Mgastric cancer xenograft model by administration with afucosylatedαFGFR2bA. (A) SCID mice bearing subcutaneous OCUM-2M xenografts weretreated with afucosylated αFGFR2bA (5 mg/kg), paclitaxel (12 mg/kg), ora combination of the two when average tumor size reached approximately285 mm³. Combining afucosylated αFGFR2bA with paclitaxel reduced tumorsize compared to either αFGFR2bA or paclitaxel alone. (B) CombinedαFGFR2bA/paclitaxel therapy significantly reduced final OCUM-2M tumorsize compared to either paclitaxel (p=0.0005) or αFGFR2bA (p=0.0009)alone. Statistical significance was determined via two-tailed, unpairedt-Test.

FIG. 5 shows enhancement of cisplatin/5-FU anti-tumor activity in anOCUM-2M gastric cancer xenograft model by administration withafucosylated αFGFR2bA. (A) SCID mice bearing subcutaneous OCUM-2Mxenografts were treated with afucosylated αFGFR2bA (5 mg/kg),fluorouracil (5-FU) plus cisplatin (2.3 and 33 mg/kg, respectively), ora combination of the three when average tumor size reached approximately260 mm³. Combining afucosylated αFGFR2bA with 5-FU/cisplatin reducedtumor size compared to either αFGFR2bA or 5-FU/cisplatin alone. (B)Combined αFGFR2bA/5-FU/cisplatin therapy significantly reduced finalOCUM-2M tumor size compared to 5-FU/cisplatin (p=0.0003). Increasedreduction in tumor size by combined αFGFR2bA/5-FU/cisplatin therapycompared to αFGFR2bA alone was notable but not statisticallysignificant. Statistical significance was determined via two-tailed,unpaired t-Test.

FIG. 6 shows efficacy of αFGFR2bA in an MFM-223 human breast cancerxenograft model. (A) MFM-223, a human breast carcinoma cell line withFGFR2 amplification, was implanted subcutaneously into SCID mice, andafucosylated αFGFR2bA therapy was initiated when average tumor sizereached approximately 80 mm³. Afucosylated αFGFR2bA (5 mg/kg)dramatically reduced growth of MFM-223 tumors compared to albumincontrol (5 mg/kg). (B) Afucosylated αFGFR2bA significantly reduced finalMFM-223 tumor size compared to albumin control (p=0.0008). Statisticalsignificance was determined via two-tailed, unpaired t-Test.

Example 5 Afucosylated Anti-FGFR2b Antibody Mediates Greater ADCC thanFucosylated Anti-FGFR2b Antibody

In vitro assays to determine the ADCC activity of αFGFR2bA antibodyversus αFGFR2bF antibody were performed. Ba/F3 FGFR2IIIb-expressingtarget cells were generated as described before. OCUM-2M cells wereobtained from Osaka City University, Osaka, Japan. All cells werecultured using standard methods. Freshly isolated PMBC from healthydonors were obtained from AllCells, Emeryville, Calif.

ADCC assays were conducted using freshly isolated human PBMCs aseffector cells at an effector to target (E:T) cell ratio of 25:1. Thetarget cells were incubated for 16 hours in the presence of effectorsand increasing concentrations of antibody. All conditions were tested intriplicate. Cytotoxicity was determined by quantifying LDH release asper the manufacturer's recommendations (Promega's CytoToxNon-Radioactive Cytotoxicity Assay). For the OCUM-2M cells, maximallysis was determined in the presence of 5% Triton X-100, and spontaneousrelease was determined in the absence of antibody. Percentage ofspecific lysis was calculated as a percentage of maximal lysis lessspontaneous release, as follows: (experimental−spontaneousrelease)/(maximal−spontaneous release)×100=% specific lysis. GraphPadsoftware curve-fitting analysis was used to obtain EC50 values.

The results for the Ba/F3 cells are shown in FIG. 9. AfucosylatedαFGFR2bA antibody showed higher potency (greater ADCC activity at lowerconcentrations) than fucosylated αFGFR2bF antibody inFGFR2IIIb-expressing Ba/F3 cells. Similar results are shown for OCUM-2Mcells in FIG. 10. Table 4 shows the potency fold increase ofafucosylated anti-FGFRb antibody compared to fucosylated anti-FGFR2bantibody in the two different cell lines shown in FIGS. 9 and 10.

TABLE 4 Potency fold increase of afucosylated versus fucosylatedanti-FGFR2b antibody EC50 (ng/mL) Potency Fold Increase of CellsαFGFR2bA αFGFR2bF αFGFR2bA OCUM-2M 5 30 6 BaF3 2 16 8 FGFR2b

Example 6 Immunohistochemical Detection of FGFR2IIIb Protein in TumorTissue

Murine αFGFR2b antibody comprising the murine variable regions ofGAL-FR21 (see U.S. Pat. No. 8,101,723 B2) and a murine IgG2a constantregion was used to detect FGFR2IIIb protein in formalin-fixed paraffinembedded (FFPE) gastric tumor tissue. FFPE tumor tissue asde-paraffinized with successive washes of xylenes and decreasingconcentrations of ethanol, and rehydrated with water for five minutes.Slide-mounted tissue sections were immersed in 0.1 mM citrate buffer (pH6.0) heated to 95°-99° C. for 15 minutes. Tissue sections were cooledand contacted with 3% H₂O₂ for 5 minutes at room temperature, and thenwashed twice in TBST (0.05M Tris, 0.15M NaCl, 0.05% Tween 20) andblocked in blocking buffer (2.5% normal horse serum in TBST) for 30minutes at room temperature. Tissue sections were then incubated with 5μg/ml αFGFR2b antibody in blocking buffer for 30 minutes at roomtemperature. Incubation times between 30 minutes and 2 hours producedsimilar staining intensities. Following three washes with TBST buffer,tissue sections were incubated with ready-to-use (RTU) biotinylatedsecondary horse anti-mouse antibody (10 μg/ml diluted in blocking buffer(Vector Laboratories, Burlingame, Calif.; Cat. No. PK-7200) for 30minutes at room temperature. Tissue sections were washed twice with TBSTbuffer, and the incubated with RTU streptavidin-horse radish peroxidase(HRP; Vector Laboratories, Cat. No. PK-7200) for 30 minutes at roomtemperature. Detection was performed using 3′,3′-diaminobenzidine (DAB)substrate (Vector Laboratories, Cat. No. PK-4105) for ten minutes atroom temperature per manufacturer's instructions. Tissue sections werethen counter-stained with hematoxylin (Dako North America, Carpinteria,Calif., Cat. No. K-8008) per manufacturer's instructions. Tissuesections were dehydrated using successive washes with increasingconcentrations of ethanol and xylenes, and then covered with acoverslip.

Staining concentrations of 0.1 to 5 mg/ml αFGFR2b antibody were found tobe particularly effective for protein detection.

The staining was scored as follows:

-   -   0 No staining with αFGFR2b antibody observed    -   1+ Faint staining is observed in samples stained with the        αFGFR2b antibody. The staining is absent in corresponding        sections stained with the negative control antibody.    -   2+ Membrane-cytoplasmic staining specific to αFGFR2b antibody is        more prevalent in the sections.    -   3+ Strong specific staining with αFGFR2b antibody with distinct        membrane localization in at least a subset of stained tumor        cells.

FIG. 11 shows exemplary 0 (A), 1+(B, C), 2+(D), and 3+(E, F) staining oftumor tissues. In FIG. 11A, ductal cells and interspersed tumor cellsshow an absence of staining using the αFGFR2b antibody. In FIG. 11B,which shows an interstitial type gastric tumor sample, tumor cells(arrows) show some staining using the αFGFR2b antibody, whilesurrounding ductal cells show no staining. In FIG. 11C, which shows adiffuse-type gastric tumor sample, tumor cells (arrows) show somestaining using the αFGFR2b antibody, while surrounding stromal cells(arrowheads) show no staining. In FIG. 11D, tumor cells (arrows) showintermediate staining using the αFGFR2b antibody, while surroundingstromal cells (arrowheads) show no staining. In FIG. 11E, tumor cells(arrows) show high membrane-localized staining using the αFGFR2bantibody, while surrounding stromal cells (arrowheads) show no staining.In FIG. 11F, tumor cells (arrows) show high membrane/cytoplasmicstaining using the αFGFR2b antibody, while surrounding stromal cells(arrowheads) show no staining.

TABLE OF SEQUENCES SEQ ID NO Description Sequence  1 Mature humanRPSFSLVED TTLEPEEPPT KYQISQPEVY VAAPGESLEV FGFR2IIIbRCLLKDAAVI SWTKDGVHLG PNNRTVLIGE YLQIKGATPRDSGLYACTAS RTVDSETWYF MVNVTDAISS GDDEDDTDGAEDFVSENSNN KRAPYWTNTE KMEKRLHAVP AANTVKFRCPAGGNPMPTMR WLKNGKEFKQ EHRIGGYKVR NQHWSLIMESVVPSDKGNYT CVVENEYGSI NHTYHLDVVE RSPHRPILQAGLPANASTVV GGDVEFVCKV YSDAQPHIQW IKHVEKNGSKYGPDGLPYLK VLKHSGINSS NAEVLALFNV TEADAGEYICKVSNYIGQAN QSAWLTVLPK QQAPGREKEI TASPDYLEIAIYCIGVFLIA CMVVTVILCR MKNTTKKPDF SSQPAVHKLTKRIPLRRQVT VSAESSSSMN SNTPLVRITT RLSSTADTPMLAGVSEYELP EDPKWEFPRD KLTLGKPLGE GCFGQVVMAEAVGIDKDKPK EAVTVAVKML KDDATEKDLS DLVSEMEMMKMIGKHKNIIN LLGACTQDGP LYVIVEYASK GNLREYLRARRPPGMEYSYD INRVPEEQMT FKDLVSCTYQ LARGMEYLASQKCIHRDLAA RNVLVTENNV MKIADFGLAR DINNIDYYKKTTNGRLPVKW MAPEALFDRV YTHQSDVWSF GVLMWEIFTLGGSPYPGIPV EELFKLLKEG HRMDKPANCT NELYMMMRDCWHAVPSQRPT FKQLVEDLDR ILTLTTNEEY LDLSQPLEQYSPSYPDTRSS CSSGDDSVFS PDPMPYEPCL PQYPHINGSV KT  2 αFGFR2b heavyQVQLVQSGAE VKKPGSSVKV SCKASGYIFT TYNVHWVRQA chain; Asn297PGQGLEWIGS IYPDNGDTSY NQNFKGRATI TADKSTSTAY is in bold andMELSSLRSED TAVYYCARGD FAYWGQGTLV TVSSASTKGP underlinedSVFPLAPSSK STSGGTAALG CLVKDYFPEP VTVSWNSGALTSGVHTFPAV LQSSGLYSLS SVVTVPSSSL GTQTYICNVNHKPSNTKVDK RVEPKSCDKT HTCPPCPAPE LLGGPSVFLFPPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVEVHNAKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKVSNKALPAPIE KTISKAKGQP REPQVYTLPP SREEMTKNQVSLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGSFFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK  3 αFGFR2b lightDIQMTQSPSS LSASVGDRVT ITCKASQGVS NDVAWYQQKP chainGKAPKLLIYS ASYRYTGVPS RFSGSGSGTD FTFTISSLQPEDIATYYCQQ HSTTPYTFGQ GTKLEIKRTV AAPSVFIFPPSDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC  4αFGFR2b heavy QVQLVQSGAE VKKPGSSVKV SCKASGYIFT TYNVHWVRQA chain variablePGQGLEWIGS IYPDNGDTSY NQNFKGRATI TADKSTSTAY regionMELSSLRSED TAVYYCARGD FAYWGQGTLV TVSS  5 αFGFR2b lightDIQMTQSPSS LSASVGDRVT ITCKASQGVS NDVAWYQQKP chain variableGKAPKLLIYS ASYRYTGVPS RFSGSGSGTD FTFTISSLQP regionEDIATYYCQQ HSTTPYTFGQ GTKLEIK  6 αFGFR2b heavy TYNVH chain (HC) HVR1  7αFGFR2b HC SIYPDNGDTS YNQNFKG HVR2  8 αFGFR2b HC GDFAY HVR3  9αFGFR2b light KASQGVSNDV A chain (LC) HVR1 10 αFGFR2b LC SASYRYT HVR2 11αFGFR2b LC QQHSTTPYT HVR3 12 Mature humanRPSFSLVED TTLEPEEPPT KYQISQPEVY VAAPGESLEV FGFR2IIIcRCLLKDAAVI SWTKDGVHLG PNNRTVLIGE YLQIKGATPRDSGLYACTAS RTVDSETWYF MVNVTDAISS GDDEDDTDGAEDFVSENSNN KRAPYWTNTE KMEKRLHAVP AANTVKFRCPAGGNPMPTMR WLKNGKEFKQ EHRIGGYKVR NQHWSLIMESVVPSDKGNYT CVVENEYGSI NHTYHLDVVE RSPHRPILQAGLPANASTVV GGDVEFVCKV YSDAQPHIQW IKHVEKNGSKYGPDGLPYLK VLKAAGVNTT DKEIEVLYIR NVTFEDAGEYTCLAGNSIGI SFHSAWLTVL PAPGREKEIT ASPDYLEIAIYCIGVFLIAC MVVTVILCRM KNTTKKPDFS SQPAVHKLTKRIPLRRQVTV SAESSSSMNS NTPLVRITTR LSSTADTPMLAGVSEYELPE DPKWEFPRDK LTLGKPLGEG CFGQVVMAEAVGIDKDKPKE AVTVAVKMLK DDATEKDLSD LVSEMEMMKMIGKHKNIINL LGACTQDGPL YVIVEYASKG NLREYLRARRPPGMEYSYDI NRVPEEQMTF KDLVSCTYQL ARGMEYLASQKCIHRDLAAR NVLVTENNVM KIADFGLARD INNIDYYKKTTNGRLPVKWM APEALFDRVY THQSDVWSFG VLMWEIFTLGGSPYPGIPVE ELFKLLKEGH RMDKPANCTN ELYMMMRDCWHAVPSQRPTF KQLVEDLDRI LTLTTNEEYL DLSQPLEQYSPSYPDTRSSC SSGDDSVFSP DPMPYEPCLP QYPHINGSVK T

1.-32. (canceled)
 33. A method of treating cancer in an individualcomprising administering to an individual with cancer an effectiveamount of a composition comprising a plurality of anti-FGFR2IIIbantibodies, wherein each anti-FGFR2IIIb antibody comprises heavy chainand light chain variable regions, wherein the heavy chain variableregion comprises: (i) HVR-H1 comprising the amino acid sequence of SEQID NO: 6; (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:7; and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 8;and the light chain variable region comprises: (iv) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO: 9; (v) HVR-L2 comprising the aminoacid sequence of SEQ ID NO: 10; and (vi) HVR-L3 comprising the aminoacid sequence of SEQ ID NO: 11; wherein at least 95% of theanti-FGFR2IIIb antibodies in the composition are afucosylated.
 34. Themethod of claim 33, wherein the cancer is selected from gastric cancer,breast cancer, ovarian cancer, endometrial cancer, pancreatic cancer,and esophageal cancer.
 35. The method of claim 34, wherein the cancer isgastric cancer.
 36. The method of claim 33, wherein the canceroverexpresses FGFR2IIIb.
 37. The method of claim 36, wherein FGFR2IIIbexpression is determined by immunohistochemistry (IHC).
 38. The methodof claim 36, wherein the cancer does not comprise FGFR2 geneamplification.
 39. The method of claim 33, wherein the cancer comprisesan FGFR2 gene amplification.
 40. The method of claim 33, wherein themethod further comprises administering at least one additionaltherapeutic agent selected from a platinum agent, paclitaxel, ABRAXANE®,docetaxel, gemcitabine, capecitabine, irinotecan, epirubicin, FOLFOX,FOLFIRI, leucovorin, fluorouracil, mitomycin C, and doxorubicinhydrochloride.
 41. The method of claim 40, wherein the platinum agent isselected from cisplatin, oxaliplatin, and carboplatin.
 42. The method ofclaim 41, wherein the additional therapeutic agent is paclitaxel. 43.The method of claim 41, wherein the additional therapeutic agent iscisplatin and/or 5-FU.
 44. The method of claim 33, wherein theanti-FGFR2IIIb antibodies comprise a heavy chain variable domaincomprising the amino acid sequence of SEQ ID NO:4 and a light chainvariable domain comprising the amino acid sequence of SEQ ID NO:
 5. 45.The method of claim 33, wherein the anti-FGFR2IIIb antibodies comprise aheavy chain comprising the amino acid sequence of SEQ ID NO: 2 and alight chain comprising the amino acid sequence of SEQ ID NO:
 3. 46. Themethod of claim 33, wherein the anti-FGFR2IIIb antibodies are monoclonalantibodies.
 47. The method of claim 33, wherein the anti-FGFR2IIIbantibodies are chimeric antibodies or humanized antibodies.
 48. Themethod of claim 33, wherein the anti-FGFR2IIIb antibodies lack fucose atAsn297.
 49. The method of claim 33, wherein the anti-FGFR2IIIbantibodies comprise a κ light chain constant region, an IgG1 heavy chainconstant region, or a κ light chain constant region and an IgG1 heavychain constant region.
 50. The method of claim 33, wherein theafucosylated antibodies have enhanced ADCC activity in vitro compared toa fucosylated anti-FGFR2IIIb antibody having the same amino acidsequence.
 51. The method of claim 50, wherein the afucosylatedanti-FGFR2IIIb antibodies cause specific lysis that is at least 30percentage points greater than specific lysis with the fucosylatedanti-FGFR2IIIb antibody.
 52. The method of claim 51, wherein ADCCactivity is determined using Ba/F3 cells expressing FGFR2IIIb as targetcells and isolated human PBMCs as effector cells.
 53. The method ofclaim 33, wherein the afucosylated antibodies have enhanced affinity forFc gamma RIIIA compared to a fucosylated anti-FGFR2IIIb antibody havingthe same amino acid sequence.
 54. The method of claim 53, wherein theafucosylated anti-FGFR2IIIb antibodies bind to Fc gamma RIIIA with atleast 3-fold greater affinity than the fucosylated anti-FGFR2IIIbantibody.
 55. The method of claim 54, wherein affinity for Fc gammaRIIIA is determined using surface plasmon resonance.
 56. The method ofclaim 55, wherein Fc gamma RIIIA is selected from Fc gamma RIIIA(V158)and Fc gamma RIIIA(F158).
 57. The method of claim 33, wherein theafucosylated anti-FGFR2IIIb antibodies bind FGFR2IIIb but do not bind toFGFR2IIIc.